Inhibitors of hiv replication

ABSTRACT

The present invention relates to novel 2,3,4-substituted 5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine compounds and pharmaceutically acceptable salts thereof, to compositions containing such compounds and to the use of such compounds as inhibitors of HIV replication.

This application is filed pursuant to 35 U.S.C. §111(a) as a UnitedStates Continuation application which claims priority to U.S.Continuation application Ser. No. 14/322,134 filed 2 Jul. 2014,presently allowed, which claims priority to U.S. Continuationapplication Ser. No. 13/295,167 filed 14 Nov. 2011, now U.S. Pat. No.8,809,363 issued 19 Aug. 2014, which claims priority to U.S. ProvisionalApplication Ser. No. 61/485,355 filed 12 May 2011, and to U.S.Provisional Application Ser. No. 61/413,618 filed 15 Nov. 2010, and theentire contents of each of the foregoing applications are incorporatedherein by reference.

The present invention is directed to 2,3,4-substituted5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine compounds andpharmaceutically acceptable salts thereof and their use as inhibitors ofthe replication of human immunodeficiency virus (HIV). The compounds ofthe present invention are useful for directly or indirectly inhibitingthe activity of one or more HIV proteins and for treating diseases orconditions mediated by HIV such as, for example, acquired immunedeficiency syndrome (AIDS). Whilst not wishing to be bound by anyspecific theory, it is believed that the compounds of the presentinvention inhibit HIV replication by direct or indirect inhibition ofthe interaction between the HIV integrase enzyme and endogenous lensepithelium-derived growth factor (LEDGF). For a discussion of thismechanism as a possible target for HIV therapy, see Llano, M et. al.Science, 314, 461-464 (2006).

WO2010/130842 discloses thieno[2,3-b]pyridine derivatives havingantiviral activity, more specifically HIV replication inhibitingproperties.

Despite the large amount of research already performed in this area,there is still a stringent need in the art for potent inhibitors of HIV.Therefore a goal of the present invention is to satisfy this urgent needby identifying efficient pharmaceutically active ingredients that areactive against HIV, less toxic, more stable (i.e. chemically stable andmetabolically stable), effective against viruses resistant to currentlyavailable drugs and/or which are more resistant to virus mutations thanexisting antiviral drugs and that can be useful, either alone or incombination with other active ingredients, for the treatment ofretroviral infections, in particular lentiviral infections, and moreparticularly HIV infections, in mammals and more specifically in humans.It is also known to those skilled in the art that the physicochemicalproperties of known drugs as well as their ADME-Tox (administration,distribution, metabolism, excretion and toxicology) properties may limitor prohibit their use in the treatment of diseases. Therefore, problemswith existing drugs which must preferably be overcome can be selectedfrom poor or inadequate physicochemical or ADME-Tox properties such assolubility, Log P, CYP inhibition, hepatic stability, and plasmastability. In particular, it would be advantageous to provide a compoundwith pharmacokinetic (PK) properties that make it suitable foronce-a-day dosing, i.e. providing the right balance of absorption,metabolism and excretion properties to achieve an exposure profile whichis consistent with once-a-day dosing. Furthermore, another goal of thepresent invention is to complement existing antiviral drugs in such away that the resulting drug combination has improved activity orimproved resistance to virus mutation than each of the individualcompounds. Once again, it would be advantageous that such a combinationwould provide a regimen suitable for once-a-day dosing.

In a first aspect, the present invention provides a compound selectedfrom

or a pharmaceutically acceptable salt thereof.

In a further embodiment of the first aspect, the present inventionprovides a compound selected from

or a pharmaceutically acceptable salt thereof.

In a further embodiment of the first aspect, the present inventionprovides a compound of formula (I)

wherein:R¹ is CH₃, CH₂CH₃, Cl, Br, CHF₂ or CF₃;

R² is H, OH or F; X is CH₂ or O;

provided that when R¹ is CH₃ and R² is H, X is O;or a pharmaceutically acceptable salt thereof.

In a further embodiment of the first aspect, the present inventionprovides a compound selected from:

or a pharmaceutically acceptable salt thereof.

In a further embodiment of the first aspect, the present inventionprovides a compound of formula (Ia)

wherein:R¹ is CH₃, CH₂CH₃, Cl, Br, CHF₂ or CF₃;

R² is H, OH or F; X is CH₂ or O;

provided that when R¹ is CH₃ and R² is H, X is O;or a pharmaceutically acceptable salt thereof.

In a further embodiment of the first aspect, the present inventionprovides a compound selected from:

or a pharmaceutically acceptable salt thereof.

In a further embodiment of the first aspect, the present inventionprovides a compound selected from:

or a pharmaceutically acceptable salt thereof.

Pharmaceutically acceptable salts of the compounds described aboveinclude the acid addition and base salts thereof.

Suitable acid addition salts may be formed from acids which formnon-toxic salts. Examples may include the acetate, adipate, aspartate,benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate,camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate,gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate,hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide,isethionate, lactate, malate, maleate, malonate, mesylate,methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate,oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogenphosphate, pyroglutamate, saccharate, stearate, succinate, tannate,tartrate, tosylate, trifluoroacetate and xinofoate salts.

Suitable base salts may be formed from bases which form non-toxic salts.Examples may include the aluminium, arginine, benzathine, calcium,choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine,olamine, potassium, sodium, tromethamine and zinc salts.

Hemisalts of acids and bases may also be formed, for example,hemisulphate and hemicalcium salts.

For a review on suitable salts, see “Handbook of Pharmaceutical Salts:Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH,Weinheim, Germany, 2002).

The compounds of the invention may exist in a continuum of solid statesranging from fully amorphous to fully crystalline. The term ‘amorphous’refers to a state in which the material lacks long range order at themolecular level and, depending upon temperature, may exhibit thephysical properties of a solid or a liquid. Typically such materials donot give distinctive X-ray diffraction patterns and, while exhibitingthe properties of a solid, are more formally described as a liquid. Uponheating, a change from solid to liquid properties occurs which ischaracterised by a change of state, typically second order (‘glasstransition’). The term ‘crystalline’ refers to a solid phase in whichthe material has a regular ordered internal structure at the molecularlevel and gives a distinctive X-ray diffraction pattern with definedpeaks. Such materials when heated sufficiently will also exhibit theproperties of a liquid, but the change from solid to liquid ischaracterised by a phase change, typically first order (‘meltingpoint’).

The compounds of the invention may exist in both unsolvated and solvatedforms. The term ‘solvate’ is used herein to describe a molecular complexcomprising the compound of the invention and a stoichiometric amount ofone or more pharmaceutically acceptable solvent molecules, for example,ethanol. The term ‘hydrate’ is employed when said solvent is water. Acurrently accepted classification system for organic hydrates is onethat defines isolated site, channel, or metal-ion coordinatedhydrates—see “Polymorphism in Pharmaceutical Solids” by K. R. Morris(Ed. H. G. Brittain, Marcel Dekker, 1995). Isolated site hydrates areones in which the water molecules are isolated from direct contact witheach other by intervening organic molecules. In channel hydrates, thewater molecules lie in lattice channels where they are next to otherwater molecules. In metal-ion coordinated hydrates, the water moleculesare bonded to the metal ion. When the solvent or water is tightly bound,the complex will have a well-defined stoichiometry independent ofhumidity. When, however, the solvent or water is weakly bound, as inchannel solvates and hygroscopic compounds, the water/solvent contentwill be dependent on humidity and drying conditions. In such cases,non-stoichiometry will be the norm.

Also included within the scope of the invention are multi-componentcomplexes (other than salts and solvates) wherein the drug and at leastone other component are present in stoichiometric or non-stoichiometricamounts. Complexes of this type include clathrates (drug-host inclusioncomplexes) and co-crystals. The latter are typically defined ascrystalline complexes of neutral molecular constituents which are boundtogether through non-covalent interactions, but could also be a complexof a neutral molecule with a salt. Co-crystals may be prepared by meltcrystallisation, by recrystallisation from solvents, or by physicallygrinding the components together—see Chem Commun, 17, 1889-1896, by O.Almarsson and M. J. Zaworotko (2004). For a general review ofmulti-component complexes, see J Pharm Sci, 64 (8), 1269-1288, byHaleblian (August 1975).

The compounds of the invention may also exist in a mesomorphic state(mesophase or liquid crystal) when subjected to suitable conditions. Themesomorphic state is intermediate between the true crystalline state andthe true liquid state (either melt or solution). Mesomorphism arising asthe result of a change in temperature is described as ‘thermotropic’ andthat resulting from the addition of a second component, such as water oranother solvent, is described as ‘lyotropic’. Compounds that have thepotential to form lyotropic mesophases are described as ‘amphiphilic’and consist of molecules which possess an ionic (such as —COO⁻ Na⁺,—COO⁻K⁺, or —SO₃ ⁻Na⁺) or non-ionic (such as —N⁻N⁺(CH₃)₃) polar headgroup. For more information, see “Crystals and the PolarizingMicroscope” by N. H. Hartshorne and A. Stuart, 4^(th) Edition (EdwardArnold, 1970).

Hereinafter all references to a compound include references to salts,solvates, polymorphs, crystal habits, multi-component complexes andliquid crystals thereof and to solvates, polymorphs, crystal habits,multi-component complexes and liquid crystals of salts thereof.

The compounds of the present invention have a chiral centre adjacent tothe carboxyl group. Thus, compounds which do not also exhibitatropisomerism (described in more detail below), may exist as twostereoisomers (i.e. enantiomers). For example:

When the 4-substituent is not symmetrical about the plane of the bond atthe 4-position, atropisomerism may also arise. This is because thearomatic ring of the 4-substituent and the pyridine portion of the5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine lie more or lessorthogonal to one another and rotation about the bond at the 4-positionof the 2,3,4-substituted 5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridinecompounds of the present invention may be restricted. Such compounds maytherefore exist as four stereoisomers (i.e. diastereoisomers). Forexample:

Compounds of formula (I) also contain aromatic moieties, such as theimidazole rings, wherein tautomeric isomerism (‘tautomerism’) can occur.This can take the form of proton tautomerism (for example in theimidazole rings) as well as valence tautomerism (for example in theother aromatic moieties). It follows that a single compound may exhibitmore than one type of isomerism.

Included within the scope of the claimed compounds of the presentinvention are all stereoisomers and tautomeric forms of the compounds,including compounds exhibiting more than one type of isomerism, andmixtures of one or more thereof.

Where a compound or formula is depicted with a specified stereochemistryat one or more chiral centres, this is intended to specify that thecompound or formula has a stereoisomeric excess of at least 80% (i.e. atleast 90% of the specified isomer and at most 10% of the other possibleisomers), preferably at least 90%, more preferably at least 94% and mostpreferably at least 99%.

Conventional techniques for the preparation/isolation of individualenantiomers/diastereoisomers include chiral synthesis from a suitableoptically pure precursor or resolution of the racemate (or the racemateof a salt or derivative) using, for example, chiral high pressure liquidchromatography (HPLC). Alternatively, the racemate (or a racemicprecursor) may be reacted with a suitable optically active compound, forexample, an alcohol, or, in the case where the compound of formula (I)contains an acidic or basic moiety, an acid or base such as tartaricacid or 1-phenylethylamine. The resulting diastereomeric mixture may beseparated by chromatography and/or fractional crystallization and one orboth of the diastereoisomers converted to the corresponding pureenantiomer(s) by means well known to a skilled person.

Chiral compounds of the invention (and chiral precursors thereof) may beobtained in enantiomerically-enriched form using chromatography,typically HPLC, on a resin with an asymmetric stationary phase and witha mobile phase consisting of a hydrocarbon, typically heptane or hexane,containing from 0 to 50% isopropanol, typically from 2 to 20%, and from0 to 5% of an alkylamine, typically 0.1% diethylamine. Concentration ofthe eluant affords the enriched mixture. Alternatively, separation canbe carried out using SFC on a resin with an asymmetric stationary phaseand with a mobile phase consisting of a gradient of CO₂ dissolved inmethanol.

Mixtures of stereoisomers may be separated by conventional techniquesknown to those skilled in the art. See, for example, “Stereochemistry ofOrganic Compounds” by E L Elie! (Wiley, New York, 1994).

The absolute configuration of a single stereoisomer of a compound of thepresent invention may be determined by solving the crystal structure ofa crystalline enzyme-compound complex using techniques known to thoseskilled in the art.

The compounds of the invention may be synthesised according to thefollowing general methods.

Coupling of a compound of formula (A) with a suitable aryl (Ar)precursor by known procedures (amination, Suzuki coupling, Negishicoupling, Stille coupling and the like) provides compounds of formula(B), which can be converted into the desired compounds of the inventionusing standard hydrolysis conditions.

Compounds of the formula (D) may also be obtained by removal of aphenolic protecting group e.g. allyl, benzyl using standard deprotectionconditions, either before or after or at the same time as hydrolysis ofthe ester group of compounds (C).

In a second aspect, the present invention provides a pharmaceuticalcomposition including a compound of the present invention or apharmaceutically acceptable salt or solvate thereof, together with apharmaceutically acceptable excipient.

The term ‘excipient’ is used herein to describe any ingredient otherthan the compound of the invention. The choice of excipient will to alarge extent depend on factors such as the particular mode ofadministration, the effect of the excipient on solubility and stability,and the nature of the dosage form.

Pharmaceutical compositions suitable for the delivery of compounds ofthe present invention and methods for their preparation will be readilyapparent to those skilled in the art. Such compositions and methods fortheir preparation may be found, for example, in “Remington'sPharmaceutical Sciences”, 19th Edition (Mack Publishing Company, 1995).

The compounds of the invention may be administered orally. Oraladministration may involve swallowing, so that the compound enters thegastrointestinal tract, or buccal or sublingual administration may beemployed by which the compound enters the blood stream directly from themouth. Formulations suitable for oral administration include both solidand liquid formulations.

Solid formulations include tablets, capsules (containing particulates,liquids, or powders), lozenges (including liquid-filled lozenges),chews, multi- and nano-particulates, gels, solid solutions, liposomalpreparations, films, ovules, and sprays.

Liquid formulations include suspensions, solutions, syrups and elixirs.Such formulations may be employed as fillers in soft or hard capsulesand typically comprise a carrier, for example, water, ethanol,polyethylene glycol, propylene glycol, methylcellulose, or a suitableoil, and one or more emulsifying agents and/or suspending agents. Liquidformulations may also be prepared by the reconstitution of a solid, forexample, from a sachet.

The compounds of the invention may also be used in fast-dissolving,fast-disintegrating dosage forms such as those described in ExpertOpinion in Therapeutic Patents, 11 (6), 981-986, by Liang and Chen(2001).

For tablet dosage forms, depending on dose, the drug may make up from 1weight % to 80 weight % of the dosage form, more typically from 5 weight% to 60 weight % of the dosage form.

In addition to the drug, tablets generally contain a disintegrant.Examples of disintegrants include sodium starch glycolate, sodiumcarboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellosesodium, crospovidone, polyvinylpyrrolidone, methyl cellulose,microcrystalline cellulose, lower alkyl-substituted hydroxypropylcellulose, starch, pregelatinised starch and sodium alginate. Generally,the disintegrant will comprise from 1 weight % to 25 weight %,preferably from 5 weight % to 20 weight % of the dosage form.

Binders are generally used to impart cohesive qualities to a tabletformulation. Suitable binders include microcrystalline cellulose,gelatin, sugars, polyethylene glycol, natural and synthetic gums,polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose andhydroxypropyl methylcellulose.

Tablets may also contain diluents, such as lactose (monohydrate,spray-dried monohydrate, anhydrous and the like), mannitol, xylitol,dextrose, sucrose, sorbitol, microcrystalline cellulose, starch anddibasic calcium phosphate dihydrate.

Tablets may also optionally comprise surface active agents, such assodium lauryl sulfate and polysorbate 80, and glidants such as silicondioxide and talc. When present, surface active agents may comprise from0.2 weight % to 5 weight % of the tablet, and glidants may comprise from0.2 weight % to 1 weight % of the tablet.

Tablets also generally contain lubricants such as magnesium stearate,calcium stearate, zinc stearate, sodium stearyl fumarate, and mixturesof magnesium stearate with sodium lauryl sulphate. Lubricants generallycomprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight %to 3 weight % of the tablet.

Other possible ingredients include anti-oxidants, colourants, flavouringagents, preservatives and taste-masking agents.

Exemplary tablets contain up to about 80% drug, from about 10 weight %to about 90 weight % binder, from about 0 weight % to about 85 weight %diluent, from about 2 weight % to about 10 weight % disintegrant, andfrom about 0.25 weight % to about 10 weight % lubricant.

Tablet blends may be compressed directly or by roller to form tablets.Tablet blends or portions of blends may alternatively be wet-, dry-, ormelt-granulated, melt congealed, or extruded before tabletting. Thefinal formulation may comprise one or more layers and may be coated oruncoated; it may even be encapsulated.

The formulation of tablets is discussed in “Pharmaceutical Dosage Forms:Tablets”, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, NewYork, 1980).

Consumable oral films are typically pliable water-soluble orwater-swellable thin film dosage forms which may be rapidly dissolvingor mucoadhesive and typically comprise a compound of formula (I), afilm-forming polymer, a binder, a solvent, a humectant, a plasticiser, astabiliser or emulsifier, a viscosity-modifying agent and a solvent.Some components of the formulation may perform more than one function.The film-forming polymer may be selected from natural polysaccharides,proteins, or synthetic hydrocolloids and is typically present in therange 0.01 to 99 weight %, more typically in the range 30 to 80 weight%. Other possible ingredients include anti-oxidants, colorants,flavourings and flavour enhancers, preservatives, salivary stimulatingagents, cooling agents, co-solvents (including oils), emollients,bulking agents, anti-foaming agents, surfactants and taste-maskingagents. Films in accordance with the invention are typically prepared byevaporative drying of thin aqueous films coated onto a peelable backingsupport or paper. This may be done in a drying oven or tunnel, typicallya combined coater dryer, or by freeze-drying or vacuuming. Solidformulations for oral administration may be formulated to be immediateand/or modified release.

Modified release formulations include delayed-, sustained-, pulsed-,controlled-, targeted and programmed release.

Suitable modified release formulations for the purposes of the inventionare described in U.S. Pat. No. 6,106,864. Details of other suitablerelease technologies such as high energy dispersions and osmotic andcoated particles are to be found in “Pharmaceutical Technology On-line”,25(2), 1-14, by Verma et al (2001). The use of chewing gum to achievecontrolled release is described in WO 00/35298.

The compounds of the invention may also be administered directly intothe blood stream, into muscle, or into an internal organ. Suitable meansfor parenteral administration include intravenous, intraarterial,intraperitoneal, intrathecal, intraventricular, intraurethral,intrasternal, intracranial, intramuscular and subcutaneous. Suitabledevices for parenteral administration include needle (includingmicroneedle) injectors, needle-free injectors and infusion techniques.

Parenteral formulations are typically aqueous solutions which maycontain excipients such as salts, carbohydrates and buffering agents(preferably to a pH of from 3 to 9), but, for some applications, theymay be more suitably formulated as a sterile non-aqueous solution or asa dried form to be used in conjunction with a suitable vehicle such assterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, forexample, by lyophilisation, may readily be accomplished using standardpharmaceutical techniques well known to those skilled in the art.

The solubility of the compound of the present invention used in thepreparation of parenteral solutions may be increased by the use ofappropriate formulation techniques, such as the incorporation ofsolubility-enhancing agents.

Formulations for parenteral administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease. Thus the compound of the invention may be formulated as asolid, semi-solid, or thixotropic liquid for administration as animplanted depot providing modified release of the active compound.Examples of such formulations include drug-coated stents andpoly(dI-lactic-coglycolic)acid (PGLA) microspheres.

The compounds of the invention may also be administered topically to theskin or mucosa, that is, dermally or transdermally. Typical formulationsfor this purpose include gels, hydrogels, lotions, solutions, creams,ointments, dusting powders, dressings, foams, films, skin patches,wafers, implants, sponges, fibres, bandages and microemulsions.Liposomes may also be used. Typical carriers include alcohol, water,mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethyleneglycol and propylene glycol. Penetration enhancers may beincorporated—see, for example, J Pharm Sci, 88 (10), 955-958, by Finninand Morgan (October 1999).

Other means of topical administration include delivery byelectroporation, iontophoresis, phonophoresis, sonophoresis andmicroneedle or needle-free (e.g. Powderject™, Bioject™, etc.) injection.

Formulations for topical administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease.

The compounds of the invention may be administered rectally orvaginally, for example, in the form of a suppository, pessary, or enema.Cocoa butter is a traditional suppository base, but various alternativesmay be used as appropriate.

Formulations for rectal/vaginal administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease.

The compounds of the invention may be combined with solublemacromolecular entities, such as cyclodextrin and suitable derivativesthereof or polyethylene glycol-containing polymers, in order to improvetheir solubility, dissolution rate, taste-masking, bioavailabilityand/or stability for use in any of the aforementioned modes ofadministration.

Drug-cyclodextrin complexes, for example, are found to be generallyuseful for most dosage forms and administration routes. Both inclusionand non-inclusion complexes may be used. As an alternative to directcomplexation with the drug, the cyclodextrin may be used as an auxiliaryadditive, i.e. as a carrier, diluent, or solubiliser. Most commonly usedfor these purposes are alpha-, beta- and gamma-cyclodextrins, examplesof which may be found in International Patent Applications Nos. WO91/11172, WO 94/02518 and WO 98/55148.

In a third aspect, the present invention provides a compound of thepresent invention or a pharmaceutically acceptable salt thereof, for useas a medicament.

A specific embodiment of this aspect of the invention is a compound ofthe present invention or a pharmaceutically acceptable salt thereof, foruse in the treatment of HIV infection.

In a fourth aspect, the present invention provides the use of a compoundof the present invention or a pharmaceutically acceptable salt thereof,for the manufacture of a medicament for the treatment of HIV infection.

In a fifth aspect, the present invention provides a method of treatmentof a mammal, including a human being, to treat HIV infection, includingadministering to said mammal an effective amount of a compound of thepresent invention or a pharmaceutically acceptable salt or solvatethereof.

The term ‘treatment’ as used herein includes both preventative andcurative treatment of a disease or disorder. It also includes slowing,interrupting, controlling or stopping the progression of a disease ordisorder. It also includes preventing, curing, slowing, interrupting,controlling or stopping the symptoms of a disease or disorder.

The compound of the present invention may be administered in combinationwith one or more additional agents for the treatment of a mammal, suchas a human, that is suffering from an infection with the HIV virus, orany other disease or condition which is related to infection with theHIV virus. The agents that may be used in combination with the compoundsof the present invention include, but are not limited to, those usefulas HIV protease inhibitors, HIV reverse transcriptase inhibitors,non-nucleoside HIV reverse transcriptase inhibitors, HIV integraseinhibitors, CCR5 inhibitors, HIV fusion inhibitors or other inhibitorsof HIV entry, maturation inhibitors, agents that act to perturb HIVcapsid multimerisation or viral core stability, compounds targeting hostproteins required for viral replication or immune evasion (such as butnot limited to PSIP1), compounds useful as immunomodulators, compoundsthat inhibit the HIV virus by an unknown mechanism, compounds useful forthe treatment of herpes viruses, compounds useful as anti-infectives,and others as described below.

Compounds useful as HIV protease inhibitors that may be used incombination with the compound of the present invention include, but arenot limited to, 141 W94 (amprenavir), CGP-73547, CGP-61755, DMP-450(mozenavir), nelfinavir, ritonavir, saquinavir (invirase), lopinavir,TMC-126, atazanavir, palinavir, GS-3333, KN 1-413, KNI-272, LG-71350,CGP-61755, PD 173606, PD 177298, PD 178390, PD 178392, U-140690,ABT-378, DMP-450, AG-1776, MK-944, VX-478, indinavir, tipranavir,TMC-114 (darunavir), DPC-681, DPC-684, fosamprenavir calcium (Lexiva),benzenesulfonamide derivatives disclosed in WO 03053435, R-944,Ro-03-34649, VX-385 (brecanavir), GS-224338, OPT-TL3, PL-100, SM-309515,AG-148, DG-35-VIII, DMP-850, GW-5950X, KNI-1039, L-756423, LB-71262,LP-130, RS-344, SE-063, UIC-94-003, Vb-19038, A-77003, BMS-182193,BMS-186318, SM-309515, JE-2147, GS-9005, telinavir (SC-52151), BILA-2185BS, DG-17, PPL-100, A-80987, GS-8374, DMP-323, U-103017, CGP-57813, andCGP-53437.

Compounds useful as inhibitors of the HIV reverse transcriptase enzymethat may be used in combination with the compound of the presentinvention include, but are not limited to, abacavir, emtricitabine(FTC), GS-840 (adefovir), lamivudine, adefovir dipivoxil,beta-fluoro-ddA, zalcitabine, didanosine, stavudine, zidovudine,tenofovir, tenofovir disoproxil fumarate, amdoxovir, SPD-754(apricitabine), SPD-756, racivir, reverset (DPC-817), MIV-210 (FLG),beta-L-Fd4C (ACH-126443, elvucitabine), MIV-310 (alovudine, FLT), dOTC,DAPD, entecavir, GS-7340, stampidine, D-d4FC (dexelvucitabine),phospahzide, fozivudine tidoxil, and fosalvudine tidoxil.

Compounds useful as non-nucleoside inhibitors of the HIV reversetranscriptase enzyme that may be used in combination with the compoundof the present invention include, but are not limited to, efavirenz,HBY-097, nevirapine, dapivirine (TMC-120), TMC-125, etravirine,delavirdine, DPC-083, DPC-961, TMC-120, capravirine, GW-678248,GW-695634, calanolide, rilpivirine (TMC-278), loviride, emivirine(MKC-442), DPC-963, MIV-150, BILR 355 BS, VRX-840773, lersivirine(UK-453061), RDEA806, and tricyclic pyrimidinone derivatives asdisclosed in WO 03062238.

Compounds useful as CCR5 inhibitors that may be used in combination withthe compound of the present invention include, but are not limited to,TAK-779, SC-351125, SCH-D, UK-427857 (maraviroc), PRO-140, and GW-873140(aplaviroc, Ono-4128, AK-602), SCH-417690 (viciviroc, SCH-D), INCB-9471,INCB-15050, TBR-220 (TAK-220), CCR5 mAb004. Other compounds useful asCCR5 inhibitors that may be used in combination with the compound of thepresent invention include, but are not limited to,(N-{(1S)-3-[3-isopropyl-5-methyl-4H-1,2,4-triazole-4-yl]-exo-8-azabicyclo[3.2.1]oct-8-yl}-1-phenylpropyl)-4,4-difluorocyclohexanecarboxamide),methyl1-endo-{8-[(3S)-3-(acetylamino)-3-(3-fluorophenyl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-2-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-5-carboxylate,andN-{(1S)-3[3-endo-(5-isobutyryl-2-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-(3-fluorophenyl)propyl}acetamide).

Compounds useful as inhibitors of HIV integrase enzyme that may be usedin combination with the compound of the present invention include, butare not limited to, raltegravir, elvitegravir (GS-9137, JTK-303),GSK-364735, MK-2048, BMS-707035, S-1360 (GW-810781), L-870810, L-870812,AR-177, BA-011, 1,5-naphthyridine-3-carboxamide derivatives disclosed inWO 03062204, compounds disclosed in WO 03047564, compounds disclosed inWO 03049690, 5-hydroxypyrimidine-4-carboxamide derivatives disclosed inWO 03035076, and L-000810810.

Fusion inhibitors for the treatment of HIV that may be used incombination with the compound of the present invention include, but arenot limited to enfuvirtide (T-20), T-1249, AMD-3100, sifuvirtide,FB-006M, TRI-1144, PRO-2000 and fused tricyclic compounds disclosed inJP 2003171381.

Maturation inhibitors for the treatment of HIV that may be used incombination with the compound of the present invention include, but arenot limited to bevirimat and vivecon.

HIV fixed drug combinations for the treatment of HIV that may be used incombination with the compound of the present invention include, but arenot limited to, combivir, atripla, trizivir, truvada, kaletra andepzicom.

CXCR4 inhibitors for the treatment of HIV that may be used incombination with the compound of the present invention include, but arenot limited to, AMD-070.

Entry inhibitors for the treatment of HIV that may be used incombination with the compound of the present invention include, but arenot limited to, SP-01A.

Gp 120 inhibitors for the treatment of HIV that may be used incombination with the compound of the present invention include, but arenot limited to, BMS-488043 and BMS-378806.

G6PD and NADH-oxidase inhibitors for the treatment of HIV that may beused in combination with the compound of the present invention include,but are not limited to, immunitin.

Other compounds that are useful inhibitors of HIV that may be used incombination with the compound of the present invention include, but arenot limited to, Soluble CD4, PRO-542, ibalizumab (TNX-355), andcompounds disclosed in JP 2003119137.

Compounds useful in the treatment or management of infection fromviruses other than HIV that may be used in combination with the compoundof the present invention include, but are not limited to, acyclovir,fomivirsen, penciclovir, HPMPC, oxetanocin G, AL-721, cidofovir,cytomegalovirus immune globin, cytovene, fomivganciclovir, famciclovir,foscarnet sodium, Isis 2922, KNI-272, valacyclovir, virazole ribavirin,valganciclovir, ME-609, PCL-016, DES6, ODN-93, ODN-112, VGV-1, ampligen,HRG-214, cytolin, VGX-410, KD-247, AMZ-0026, CYT-99007A-221, DEBIO-025,BAY 50-4798, MDX-010 (ipilimumab), PBS-119, ALG-889, PA-1050040 (PA-040)and filibuvir (PF-00868554).

Compounds that act as immunomodulators and may be used in combinationwith the compound of the present invention include, but are not limitedto, AD-439, AD-519, Alpha Interferon, AS-101, bropirimine, acemannan,CL246,738, EL10, FP-21399, gamma interferon, granulocyte macrophagecolony stimulating factor, IL-2, immune globulin intravenous, IMREG-1,IMREG-2, imuthiol diethyl dithio carbamate, alpha-2 interferon,methionine-enkephalin, MTP-PE, granulocyte colony stimulating sactor,remune, rCD4, recombinant soluble human CD4, interferon alfa-2,SK&F106528, soluble T4 yhymopentin, tumor necrosis factor (TNF),tucaresol, recombinant human interferon beta, and interferon alfa n-3.

Anti-infectives that may be used in combination with the compound of thepresent invention include, but are not limited to, atovaquone,azithromycin, clarithromycin, trimethoprim, trovafloxacin,pyrimethamine, daunorubicin, clindamycin with primaquine, pastill,ornidyl, eflornithine pentamidine, rifabutin, spiramycin,intraconazole-R51211, trimetrexate, daunorubicin, chloroquine,recombinant human erythropoietin, recombinant human growth hormone,megestrol acetate, testerone, and total enteral nutrition.

Antifungals that may be used in combination with the compound of thepresent invention include, but are not limited to, anidulafungin, C31G,caspofungin, DB-289, fluconzaole, itraconazole, ketoconazole,micafungin, posaconazole, and voriconazole.

Other compounds that may be used in combination with the compound of thepresent invention include, but are not limited to, acemannan, ansamycin,LM 427, AR177, BMS-232623, BMS-234475, Cl-1012, curdlan sulfate, dextransulfate, STOCRINE EL10, hypericin, lobucavir, novapren, peptide Toctabpeptide sequence, trisodium phosphonoformate, probucol, andRBC-CD4.

In addition, the compound of the present invention may be used incombination with anti-proliferative agents for the treatment ofconditions such as Kaposi's sarcoma. Such agents include, but are notlimited to, inhibitors of metallo-matrix proteases, A-007, bevacizumab,BMS-275291, halofuginone, interleukin-12, rituximab, paclitaxel,porfimer sodium, rebimastat, and COL-3.

Such a combination may be administered such that the compound of thepresent invention is present in the same pharmaceutical composition asthe additional agent(s) described above. Alternatively, such acombination may be administered such that the compound of the presentinvention is present in a pharmaceutical composition that is separatefrom the pharmaceutical composition in which the additional agent(s)is(are) found. If the compound of the present invention is administeredseparately from the additional agent(s), such administration may takeplace concomitantly or sequentially with an appropriate period of timein between.

Additionally, the compound of the present invention may be administeredin combination with one or more additional agents that have the effectof increasing the exposure of the mammal to the compound of theinvention. The term ‘exposure’, as used herein, refers to theconcentration of the compound of the invention in the plasma of a mammalas measured over a period of time. The exposure of a mammal to aparticular compound can be measured by administering the compound of theinvention to a mammal in an appropriate form, withdrawing plasma samplesat predetermined times, and measuring the amount of a compound of theinvention in the plasma using an appropriate analytical technique, suchas liquid chromatography or liquid chromatography/mass spectroscopy. Theamount of the compound of the invention present in the plasma at acertain time is determined and the concentration and time data from allthe samples are plotted to afford a curve. The area under this curve iscalculated and affords the exposure of the mammal to the compound.

The terms ‘exposure’, ‘area under the curve’, and ‘area under theconcentration/time curve’ are intended to have the same meaning and maybe used interchangeably.

Among the agents that may be used to increase the exposure of a mammalto a compound of the present invention are those that can act asinhibitors of at least one isoform of the cytochrome P450 (CYP450)enzymes. The isoforms of CYP450 that may be beneficially inhibitedinclude, but are not limited to, CYP1A2, CYP2D6, CYP2C9, CYP2C19 andCYP3A4. Suitable agents that may be used to inhibit CYP3A4 include, butare not limited to, ritonavir, delavirdine,N-(3,4-difluorobenzyl)-2-{[(4-methoxypyridin-3-yl)amino]sulfonyl}-N-methylbenzamide,andN-(1-(5-(4-fluorobenzyl)-3-(pyridin-4-yl)-1H-pyrazole-1-carbonyl)piperidin-4-yl)methanesulfonamide.

Such a combination may be administered such that the compound of thepresent invention is present in the same formulation as the additionalagent(s) described above. Alternatively, such a combination may beadministered such that the compound of the present invention is presentin a pharmaceutical composition that is separate from the pharmaceuticalcomposition in which the additional agent(s) is(are) found. If thecompound of the present invention is administered separately from theadditional agent(s), such administration may take place concomitantly orsequentially with an appropriate period of time in between.

Inasmuch as it may be desirable to administer a combination of activecompounds, for example, for the purpose of treating a particular diseaseor condition, it is within the scope of the present invention that twoor more pharmaceutical compositions, at least one of which contains acompound in accordance with the invention, may conveniently be combinedin the form of a kit suitable for co-administration of the compositions.

Thus the kit of the invention comprises two or more separatepharmaceutical compositions, at least one of which contains a compoundof the present invention in accordance with the invention, and means forseparately retaining said compositions, such as a container, dividedbottle, or divided foil packet. An example of such a kit is the familiarblister pack used for the packaging of tablets, capsules and the like.

The kit of the invention is particularly suitable for administeringdifferent dosage forms, for example, oral and parenteral, foradministering the separate compositions at different dosage intervals,or for titrating the separate compositions against one another. Toassist compliance, the kit typically comprises directions foradministration and may be provided with a so-called memory aid.

The following procedures illustrate methods suitable for the preparationof compounds of the present invention.

General Methods

LCMS (2 min acidic) A: 0.1% formic acid in water, B: 0.1% formic acid inacetonitrile, Column: Agilent Extend C18 phase 30×3 mm with 3 micronparticle size, Gradient: 90-0% A over 1.8 min, 0.55 min hold, 0.15 minre-equilibration, 1.6 mL/min flow rate, UV: 210 nm-450 nm DAD,Temperature: 50° C.

LCMS (5 min acidic) A: 0.1% formic acid in water B: 0.1% formic acid inacetonitrile Column: Agilent Extend C18 phase 50×3 mm with 3 micronparticle size Gradient: 95-0% A over 3.5 min, 1 min hold, 0.4 minre-equilibration, 1.2 mL/min flow rate UV: 210 nm-450 nm DADTemperature: 50° C.

LCMS (12 min acidic) A: 0.1% formic acid in water B: 0.1% formic acid inacetonitrile Column: Agilent SB C18 phase 50×3 mm with 3 micron particlesize Gradient: 95% A 1 min hold, 95-0% A over 8 min, 2.5 min hold, 0.50min re-equilibration, 1.2 mL/min flow rate UV: 210 nm-450 nm DADTemperature: 50° C. LCMS (5 min basic) A: methanol, B: 10 mM ammoniumbicarbonate in water @ pH10, Column: XBridge C18 2.1×30 mm with 5 micronparticle size, Gradient: 95-5% A over 2.9 min, 0.9 min hold, 0.1 minre-equilibration, 0.5 mL/min flow rate UV: 215-350 nm DAD Temperature25° C.

H NMR Collected on Varian Gemini 400 MHz at 30 C.

Preparation 1: Ethyl2-amino-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate

To a solution of ethyl cyanoacetate (427 mL, 4 mol) in ethanol (4 L) wasadded sulfur (153.88 g, 4.80 mol), morpholine (422 mL, 4.80 mol) andcyclohexanone (497 mL, 4.80 mol) and the resulting solution was stirredat 50° C. for 18 hours. The reaction was cooled to room temperature andfiltered to remove solids. The filter cake was washed with cold ethanoland then dried to give the title compound as a pale yellow solid, 608.4g. The mother liquors were cooled in an ice bath and the resultingprecipitate was collected by filtration. The solid was purified by dryflash chromatography eluting with ethyl acetate in heptane (20-30%) toyield a further 38.62 g of the title compound. The two solids werecombined to give 647.02 g of the title compound in a 72% yield. ¹H NMR(400 MHz, CDCl₃) δ ppm 1.34 (t, 3H), 1.73-1.75 (m, 4H), 2.46-2.49 (m,2H), 2.63-2.69 (m, 2H), 4.25 (q, 2H), 5.92 (br s, 2H).

Preparation 2: Ethyl-3-ethoxybut-2-eneoate

To a solution of ethylacetoacetate (2.7 kg, 20.74 mol) in ethanol (4 L)was added conc.H₂SO₄ (4 ml) at 25° C. under a nitrogen atmosphere. Themixture was heated to 50° C. before adding triethylorthoformate (3073.6g, 20.74 mol) drop wise. The mixture was stirred at 50° C. for 16h. Themixture was concentrated under reduced pressure to give the titlecompound (2.8 kg, 85%) as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 4.9(s, 1H), 4.1 (m, 2H), 3.7 (m, 2H), 2.2 (s, 3H), 1.2 (m, 3H), 1.1 (m,3H).

Preparation 3: Ethyl(4-hydroxy-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl)carboxylate

In a 10 L reaction vessel, pyridinium p-toluenesulfonic acid (39.7 g,158 mMol) was added to a stirred solution ofethyl-2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate(Preparation 1) and ethyl-3-ethoxybut-2-eneoate (Preparation 2) intoluene (6.0 L) at room temperature under argon. The resulting mixturewas heated to reflux, and stirred at reflux for 6 hours. The mixture wascooled 40° C. and stirred at 40° C. for 16 hours. The mixture was heatedto 50° C. (to dissolve the precipitate) and the solution was run out ofthe reaction vessel. The reaction vessel was charged with 21% (w/w)sodium ethoxide in ethanol (1.1 L, 3.48 Mol) and ethanol (2.0 L), andthe resulting mixture was heated to 50° C. The reaction mixture was thenpoured back into the reaction vessel over 5 minutes. The resultingmixture was heated to reflux and stirred at reflux for 2 hours. Themixture was cooled to 30° C. and split into 2 approximately equalbatches. Each batch was treated with Celite® (approx. 2 L) andconcentrated under reduced pressure. The resulting solids were suspendedin water (2.0 L, 40° C.) and stirred vigorously until all solids werefree flowing. The solids were collected by filtration through a pad ofCelite®, and the filter-cake was washed with water (2.5 L, 40° C.). Thefiltrate was washed with diethylether (2×1.3 L) and then cooled to 15°C. 6 M hydrochloric acid was added carefully until pH 4 was achieved.The resulting precipitate was collected by filtration and thefilter-cake was washed with dilute hydrochloric acid (500 mL). Thefilter-cake was dried in a drying oven, at 50° C. for 2 days. This gavethe title compound as a yellow solid (584 g, 64%).

Preparation 4: Ethyl4-chloro-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-carboxylate

Ethyl4-hydroxy-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-carboxylate(Preparation 3, 150 g, 515 mmol) was added portionwise to phosphorusoxychloride (450 mL, 4.92 mol) and the resulting mixture stirred at 100°C. for 1 hour and then cooled to room temperature. The volatiles wereremoved in vacuo, and the residue was carefully poured into a vigorouslystirred ice/water mixture. Ethyl acetate (500 mL) was added to themixture. The mixture was adjusted to pH 8, by addition of 10M aqueoussodium hydroxide solution. The layers were separated and the aqueouslayer was further extracted with ethyl acetate (3×500 mL). The combinedorganic extracts were washed with brine (300 mL), dried over MgSO₄ andconcentrated in vacuo. The resulting oil was poured into acrystallisation dish, where it solidified on standing to give the titlecompound, 140.69 g, in an 88% yield. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.42(t, 3H), 1.86-1.89 (m, 4H), 2.59 (s, 3H), 2.82-2.84 (m, 2H), 3.08-3.11(m, 2H), 4.46 (q, 2H).

Preparation 5: Ethyl(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl)carboxylate

Sodium iodide (994.0 g, 6.63 Mol) was added over 5 minutes to a stirredsolution of acetyl chloride (177 mL, 2.48 Mol) and ethyl(4-chloro-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl)carboxylate(Preparation 4, 257 g, 0.83 Mol) in acetonitrile (2.0 L) at roomtemperature. The resulting mixture was heated to reflux and stirred for30 hours. The mixture was allowed to cool to room temperature and stoodfor 72 hours. The solids were collected by filtration and washed withcold acetonitrile (500 mL). The solids were partitioned betweendichloromethane (1.5 L) and water (0.75 L). The aqueous layer wasbasified to pH 9 by addition of 2 M sodium hydroxide solution and thetwo layers were separated. The organic layer was washed with 2 M sodiumthiosulphate solution (800 mL), brine (800 mL), dried (Na₂SO₄), filteredand concentrated under reduced pressure to give the title compound(260.0 g, 78%) as a beige solid. This material was determined by ¹H NMRspectroscopy to be a 13:1 mixture of ethyl(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl)carboxylateand ethyl(4-chloro-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl)carboxylaterespectively.

Preparation 6:(4-Iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl)methanol

To a solution of ethyl(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl)carboxylate(Preparation 5, 260.2 g, 0.65 Mol) in CH₂Cl₂ (2.0 L) was added 25% (w/w)diisobutyl aluminium hydride (2.0 L, 1.97 Mol) at 5° C. over 1.5 hoursunder argon. The resulting mixture was allowed to warm to roomtemperature and stirred for 16 hours. The resulting mixture was dilutedwith dichloromethane (1.0 L), cooled to 0° C. and 2 M hydrochloric acid(200 mL) was added very carefully. 6M hydrochloric acid was then addeduntil the pH of the aqueous was measured at pH 2. The resulting mixturewas stirred vigorously for 1 hour. The precipitate was collected byfiltration and the filter-cake was washed with dichloromethane (300 mL)and water (300 mL). The solids were suspended in dichloromethane (250mL) and 1 M sodium hydroxide solution (400 mL) and stirred vigorouslyuntil the solids were free flowing. The solids were collected byfiltration, washing the filter-cake with water (200 mL). The filter-cakewas concentrated from propan-2-ol (500 mL), methanol (500 mL) anddichloromethane (500 mL) sequentially under reduced pressure to give thetitle compound as a cream coloured solid (212.3 g, 91%).

Preparation 7:(4-Iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl)carbaldehyde

To a suspension of(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl)methanol(Preparation 6, 150.0 g, 0.42 Mol) in triethylamine (174 mL, 1.25 Mol)and dimethylsulfoxide (1.1 L) was added sulfur trioxide pyridinecomplex, portion-wise at 5° C. The resulting mixture was stirred at roomtemperature for 16 hours. The mixture was poured into vigorouslystirring ice water (1.0 L). The solids were collected by filtration andthe filter-cake washed with water (750 mL). The resulting solids weredissolved in dichloromethane/ethyl acetate (2.0 L, 9:1) (requiredwarming) and the solution was dried (MgSO₄), filtered and concentratedunder reduced pressure to give the title compound as a white solid(135.0 g, 91%). ¹H NMR (400 MHz, CDCl₃) δ 10.36 (s, 1H), 3.32-3.20 (m,2H), 2.89-2.79 (m, 2H), 2.76 (s, 3H) 1.95-1.83 (m, 4H).

Preparation 8:2-(4-Iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)-2-[(trimethylsilyl)oxy]acetonitrile

To a suspension of4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-carbaldehyde(Preparation 7, 115.31 g, 322.8 mmol) in dichloromethane (1.75 L) wasadded zinc iodide (41.1 g, 128.8 mmol) and the resulting mixture wascooled to 5° C. Trimethylsilyl cyanide (129 mL, 968.4 mmol) was addeddropwise over 10 minutes, and the mixture was allowed to warm to roomtemperature over 2 hours. Water (750 mL) and dichloromethane (1 L) wereadded and after stirring for 1 hour, the layers were separated. Theaqueous layer was extracted with dichloromethane (500 mL) and thecombined organic layers were washed with water (700 mL) and brine (700mL), dried over Na₂SO₄ and concentrated in vacuo to give the titlecompound, 145.61 g, in a 99% yield. ¹H NMR (400 MHz, CDCl₃) δ ppm 0.22(s, 9H), 1.82-1.93 (m, 4H), 2.56-2.86 (m, 2H), 2.92 (s, 3H), 3.18-3.25(m, 2H), 6.48 (s, 1H).

Preparation 9:Methyl-2-[4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl]-2-hydroxyacetate

To a stirred suspension of2-(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl)-2-trimethylsiloxyacetonitrile(Preparation 8, 168.0 g, 369.10 mMol) in methanol (2.5 L) was addedconcentrated sulphuric acid (410 mL, 7.38 Mol) at 5° C. The mixture washeated to 70° C. and stirred for 36 hours. After allowing the mixture tocool to room temperature, the mixture was diluted with water (1.0 L) andethyl acetate (2.0 L), and then cooled to 5° C. The pH of the aqueouslayer was adjusted to pH 8 by careful addition of 6 M aqueous sodiumhydroxide solution. This resulted in the precipitation of solids. Thesolids were collected by filtration, and the filter-cake was washed withwarm water (500 mL) and ethyl acetate (200 mL). The filtrate was savedfor further manipulation. The filter-cake was suspended in methanol (500mL) and stirred vigorously until the solid was free flowing. The solidswere collected by filtration and the filter-cake was washed withmethanol (2×200 mL) and diethylether (2×200 mL). The resulting solid wasdried at 50° C. for 16 hours. This gave the title compound (66.5 g, 43%)as a white solid. The layers of the saved filtrates were separated andthe organic layer was concentrated under reduced pressure. The resultingresidue was triturated with methanol (100 mL), ethyl acetate (100 mL)and diethylether (100 mL). The resulting solid was collected byfiltration and dried at 50° C. for 16 hours. This gave an additionalportion of the title compound (38.3 g, 25%) as a white solid.

Preparation 10: Methyltert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetate

A suspension of methylhydroxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetate(Preparation 9, 10.0 g, 23.97 mmol) in tert-butyl acetate (250 mL) wasstirred vigorously for 5 minutes prior to the dropwise addition ofperchloric acid (70%, 6.15 mL, 71.90 mmol). The resulting mixture wasstirred at room temperature for 20 minutes. The mixture was neutralisedby addition of a saturated aqueous sodium hydrogen carbonate solution(60 mL) and extracted with ethyl acetate (250 mL). The layers wereseparated and the aqueous layer was further extracted with ethyl acetate(250 mL). The combined organic layers were washed with brine (250 mL),dried over MgSO₄ and concentrated in vacuo to yield the crude product.The residue was purified by flash column chromatography eluting withethyl acetate/heptane (10-40%) to give the title compound, 4.26 g, in a38% yield. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.23 (s, 9H), 1.81-1.92 (m,4H), 2.66 (s, 3H), 2.79-2.89 (m, 2H), 3.21-3.27 (m, 2H), 3.68 (s, 3H),5.95 (s, 1H).

Preparation 11:Ethyl-2-tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetate

Ethyl-2-[4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl]-2-hydroxyacetate(3 g, 7 mmol) was taken up in 20 mL dichloromethane and tert-butylacetate (20 mL, 170 mmol) and the mixture coiled to 3° C. in anice/water bath. Concentrated sulphuric acid (1.14 mL, 21 mmol) was thenadded dropwise and the mixture allowed to warm to room temperature over3 hours. The reaction was quenched by the addition of 100 mL of 1M NaOHand 100 mL water and the organic layer separated. The organics werewashed with brine (100 mL), dried over MgSO₄ and evaporated underreduced pressure. The residue was purified by flash chrmoatography usinga gradient of EtOAc in heptane as eluant (0:100 to 30:70) to afford thetitle compound as a white solid (1.22 g, 36%). ¹H NMR (400 MHz, CDCl₃) δ1.20 (t, 3H), 1.23 (s, 9H), 1.81-1.94 (m, 4H), 2.66 (s, 3H), 2.80-2.87(m, 2H), 3.22-3.31 (m, 2H), 4.10-4.22 (m, 2H), 5.90 (s, 1H).

Preparation 12:Tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)aceticacid

To a solution of Methyltert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetate(Preparation 10, 63.95 g, 135 mmol) in tetrahydrofuran (1 L) andmethylated spirit (1 L) was dropwise added aqueous sodium hydroxide (1M, 811 mL, 811 mmol). The resulting mixture was stirred at 60° C. for 90minutes and then allowed to cool to room temperature overnight. Thevolatile solvents were removed in vacuo, and the remaining aqueousresidue was diluted with water (400 mL) and extracted with tert-butylmethyl ether (600 mL). The organic layer was washed with water (400 mL)and then cooled on ice. 2 M hydrochloric acid was added to pH 5 and theresulting solids were collected by filtration, washing with water. Thesolid was dissolved in 2-methyl tetrahydrofuran (300 mL) and stirred for10 minutes. An aqueous layer had appeared and this was separated. Theaqueous layer was extracted with 2-methyl tetrahydrofuran (2×300 mL).The combined organic layers were dried over MgSO₄ and concentrated invacuo to give the title compound as a pale yellow solid, 54.35 g, in an88% yield. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.27 (s, 9H), 1.81-1.93 (m,4H), 2.65 (s, 3H), 2.76-2.88 (m, 2H), 3.20-3.27 (m, 2H), 6.13 (s, 1H),9.66 (br s, 1H).

Preparation 13:(4R)-4-benzyl-3-[(2R)-2-tert-butoxy-2-(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetyl]-1,3-oxazolidin-2-one(13A) and(4R)-4-benzyl-3-[(2S)-2-tert-butoxy-2-(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetyl]-1,3-oxazolidin-2-one(13B)

To a solution oftert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)aceticacid (Preparation 12, 54.35 g, 118 mmol) in tetrahydrofuran (1 L) wereadded [(benzotriazol-1-yloxy)-dimethylamino-methylene]-dimethyl-ammoniumhexafluoro phosphate (67.31 g, 177 mmol) and ethyl-di-isopropyl-amine(61.8 mL, 355 mmol) and the resulting reaction mixture was stirred at40° C. for 2 hours. After 90 minutes (R)-4-benzyl-2-oxazolidinone (41.93g, 237 mmol) was dissolved in tetrahydrofuran (500 mL) and treated withsodium hydride (60% in mineral oil) and stirred for 30 minutes at roomtemperature. After this time, the two mixtures were combined and stirredat 40° C. for 8 hours. Precipitated material was collected byfiltration. Solids were partitioned between ethyl acetate (500 mL) and asaturated aqueous solution of sodium hydrogen carbonate (500 mL). Thelayers were separated and the aqueous layer was further extracted withethyl acetate (2×500 mL). The combined organic layers were washed withbrine (500 mL), dried over MgSO₄ and concentrated in vacuo to yield abrown oil, 1.2 g. The original THF filtrate was washed with a saturatedaqueous solution of sodium hydrogen carbonate (500 mL) with the additionof ethyl acetate (1 L) to enable separation of layers. The aqueous layerwas washed with ethyl acetate (2×250 mL). The combined organic extractswere washed with brine (500 mL), dried over MgSO₄ and concentrated invacuo. The crude residue was combined with the brown oil from the solidswork up and purified by dry flash column chromatography eluting with agradient of heptane and ethyl acetate (0% to 20%). Product containingfractions were concentrated in vacuo to give a pale yellow semi-solid,72 g. The residue was further purified by column chromatography elutingwith a gradient of heptane and ethyl acetate (0% to 10%) to separate thediastereomers. Both were recrystallised from methylated spirits. The toprunning spot was isolated as a white solid, 23.75 g, in a 32% yield(13A). The bottom running spot was isolated as a white solid, 21.36 g,in a 29% yield (13B). 13A: ¹H NMR (400 MHz, CDCl₃) δ ppm 1.25 (s, 9H),1.80-1.92 (m, 4H), 2.85 (s, 3H), 2.79-2.90 (m, 2H), 2.94 (dd, 1H),3.15-3.30 (m, 2H), 4.16 (dd, 1H), 4.26 (t, 1H), 4.71-4.80 (m, 1H), 6.90(s, 1H), 7.23-7.36 (m, 4H). 13B: ¹H NMR (400 MHz, CDCl₃) δ ppm 1.17 (s,9H), 1.82-1.95 (m, 4H), 2.84 (s, 3H), 2.78-2.87 (m, 3H), 3.18-3.31 (m,2H), 4.17-4.22 (m, 1H), 4.30 (t, 1H), 4.70-4.78 (m, 1H), 6.93 (s, 1H),7.17-7.33 (m, 4H).

Preparation 14:(2R)-tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)aceticacid

To a solution of(4R)-4-benzyl-3-[(2R)-2-tert-butoxy-2-(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetyl]-1,3-oxazolidin-2-one(Preparation 13A, 23.75 g, 38.40 mmol) in tetrahydrofuran (450 mL) andwater (150 mL) at 0° C. was added dropwise a solution of lithiumhydroxide hydrate (3.35 g, 80.64 mmol) and hydrogen peroxide (27%aqueous, 18.3 mL, 161.27 mmol). The resulting solution was stirred at 0°C. for 15 minutes, then for 2 hours at room temperature. A saturatedsolution of sodium sulphite (500 mL) was added followed by water (500mL), and the mixture stirred for 15 minutes. The mixture was acidifiedto pH 4 by the addition of 6 M hydrochloric acid. The mixture wasextracted with dichloromethane (4×500 mL). The combined organic layerswere dried over MgSO₄ and concentrated in vacuo. The resulting whitesolid was crystallised from methylated spirits (150 mL) with heating atreflux for 5 minutes. The mixture was allowed to cool to roomtemperature overnight. The resulting solid was collected by filtrationand washed with cold methylated spirits and dried in vacuo to give thetitle compound as a white solid, 12.01 g, in a 68% yield. ¹H NMR (400MHz, CDCl₃) δ ppm 1.27 (s, 9H), 1.81-1.93 (m, 4H), 2.65 (s, 3H),2.75-2.88 (m, 2H), 3.18-3.28 (m, 2H), 6.13 (s, 1H), 9.66 (br s, 1H).

Preparation 15:(2S)-tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)aceticacid

To a solution of(4R)-4-benzyl-3-[(2S)-2-tert-butoxy-2-(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetyl]-1,3-oxazolidin-2-one(Preparation 13B, 21.36 g, 34.53 mmol) in tetrahydrofuran (400 mL) andwater (130 mL) at 0° C. was added dropwise a solution of lithiumhydroxide hydrate (3.04 g, 72.52 mmol) and hydrogen peroxide (27%aqueous, 16.4 mL, 145.04 mmol). The resulting solution was stirred at 0°C. for 15 minutes, then for 2 hours at room temperature. A saturatedsolution of sodium sulphite (500 mL) was added followed by water (500mL), and the mixture stirred for 15 minutes. The mixture was acidifiedto pH 4 by the addition of 6 M hydrochloric acid. The mixture wasextracted with dichloromethane (4×500 mL). The combined organic layerswere dried over MgSO₄ and concentrated in vacuo. The resulting whitesolid was crystallised from methylated spirits (150 mL) with heating atreflux for 5 minutes. The mixture was allowed to cool to roomtemperature overnight. The resulting solid was collected by filtrationand washed with cold methylated spirits and dried in vacuo to give thetitle compound as a white solid, 10.25 g, in a 65% yield. ¹H NMR (400MHz, CDCl₃) δ ppm 1.27 (s, 9H), 1.81-1.93 (m, 4H), 2.65 (s, 3H),2.75-2.88 (m, 2H), 3.18-3.28 (m, 2H), 6.13 (s, 1H).

Preparation 16: Methyl(2S)-tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetate

To a solution of(2S)-tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)aceticacid (Preparation 15, 1.0 g, 2.178 mmol) in dichloromethane (34 mL) wasadded [(benzotriazol-1-yloxy)-dimethylamino-methylene]-dimethyl-ammoniumhexafluoro phosphate (1.24 g, 3.266 mmol) and ethyl-di-isopropyl-amine(600 μL, 3.266 mmol) and the resulting reaction mixture was stirred at30° C. for 2 hours. Methanol (17 mL) was added and the reaction mixturewas stirred at 30° C. for 18 hours. The reaction was cooled to roomtemperature and diluted with dichloromethane (50 mL). The solution waswashed with a saturated aqueous solution of sodium hydrogen carbonate(50 mL), water (50 mL) and brine (50 mL). The organic layer was driedover MgSO₄ and concentrated in vacuo. The residue was purified by flashchromatography eluting with 5% ethyl acetate in heptane to give thetitle compound as a white solid, 963 mg, in a 93% yield. ¹H NMR (400MHz, CDCl₃) δ ppm 1.22 (s, 9H), 1.85-1.88 (m, 4H), 2.65 (s, 3H),2.82-2.86 (m, 2H), 3.23-3.26 (m, 2H), 3.68 (s, 3H), 5.94 (s, 1H).

Preparation 17: (4-chloro-2-hydroxyphenyl)boronic acid

To a solution of (4-chloro-2-methoxyphenyl)boronic acid (1.0 g, 5.36mmol) in dichloromethane (5 mL) at 0° C. was added boron tribromide (1 Min dichloromethane, 10 mL, 10 mmol). The reaction was stirred at 0° C.for 1 hour, then allowed to warm to room temperature and stirred as suchfor 18 hours. The reaction was quenched carefully with water. Theresulting precipitate was collected by filtration, to give a whitesolid, 260 mg. The layers were separated and the organic layer was driedover Na₂SO₄ and concentrated in vacuo to give a white solid, 300 mg. Thetwo batches of solid were combined to give the title compound as a whitesolid, 560 mg, in a 61% yield. This material was taken on to Preparation18 without purification.

Preparation 18:5-chloro-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol

To a solution of (4-chloro-2-hydroxyphenyl)boronic acid (Preparation 15,200 mg, 1.16 mmol) in dichloromethane (15 mL) was added pinacol (274 mg,2.32 mmol) and the resulting solution was stirred at room temperaturefor 18 hours. The reaction mixture was washed with water (10 mL), driedover Na₂SO₄ and concentrated in vacuo to give the title compound as apale yellow solid, 280 mg, in a 95% yield.

¹H NMR (400 MHz, CDCl₃) δ ppm 1.35 (s, 12H), 6.86-6.88 (m, 2H), 7.51 (d,1H), 7.89 (s, 1H).

Preparation 19:2-(2-fluoro-4-methylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

To a solution of (2-fluoro-4-methylphenyl)boronic acid (500 mg, 3.24mmol) in diethyl ether (20 mL) was added pinacol (360 mg, 3.24 mmol) and4-toluenesulfonic acid monohydrate (30 mg, 162 μmol) and the resultingsolution was stirred at room temperature for 18 hours. The reactionmixture was washed with a saturated aqueous sodium hydrogen carbonatesolution (20 mL), dried over MgSO₄ and concentrated in vacuo to give thetitle compound as a white solid, 740 mg, in a 97% yield. ¹H NMR (400MHz, CDCl₃) δ ppm 1.33 (s, 12H), 2.34 (s, 3H), 6.82 (d, 1H), 6.93 (d,1H), 7.60 (d, 1H).

EXAMPLE 1Tert-butoxy(2-methyl-4-pyrimidin-5-yl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)aceticacid

Methyl-tert-butoxy[4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridine-3-yl]acetate(Preparation 10) may be reacted with pyrimidine boronic acid in thepresence of potassium carbonate andtetrakis(triphenylphosphine)palladium(0) to provide the methyl ester ofthe above compound. Hydrolysis with aqueous lithium hydroxide may beused to provide the final product.

Antiviral Activity >20 μM (n=2) (S8737E)

EXAMPLE 2(2S)-tert-butoxy(2-methyl-4-pyrimidin-5-yl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)aceticacid

A sample of the product from Example 1 may be dissolved in a mixture ofmethanol and dichloromethane (1:1) loaded onto a Chiralpak IC column(250×20 mm internal diameter) and eluted with methanol/CO₂ (60:40) atambient temperature and a flow rate of 60 g/min. Fractions containing asingle enantiomer may be combined and evaporated under reduced pressureto give the product enantiomer. Chiral purity may be assessed by chiralhplc using a Chiralpak IC column eluting with hexane/isopropanol.

EXAMPLE 3Ethoxy[4-(2-hydroxy-4-methylphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid

Step 1

To a solution of 1-(2-hydroxy-4-methyl-phenyl)-ethanone (10 g, 67 mmols)in dimethylformamide (100 mL) was added potassium carbonate (18.4 g, 2eq, 133 mmols), followed by allyl bromide (8.06 g, 5.75 mL, 1 eq, 67mmols). The reaction was stirred at room temperature for 16 hours. Theresidue was partitioned between ethyl acetate (200 mL) and water (800mL), the organics were separated, and washed with brine (50 mL), dried(MgSO4), filtered and concentrated in vacuo. After cooling theconcentrate to room temperature 1-(2-allyloxy-4-methyl-phenyl)-ethanonecrystallised as colourless platelets 12.40 g (98% yield). LCMS (2 minacidic) 1.20 min 72-100% pure by UV, ES+/AP+ 191. HNMR (CDCl₃) >95% pure7.68 (d, J=8.01 Hz, 1H), 6.80-6.83 (m, 1H), 6.76 (s, 1H), 6.10 (m, 1H),5.44 (dq, J=17.5, 1.5 Hz, 1H), 5.33 (dq, J=11.0 Hz, 1.5 Hz, 1H),4.61-4.66 (m, 2H), 2.63 (s, 3H), 2.37 (s, 3H).

Step 2

To 1-(2-allyloxy-4-methyl-phenyl)-ethanone (13.9 g, 73 mmols) was addeddimethylformamide dimethylacetal (56 mL, 422 mols, 5.8 eq), and thereaction was heated to reflux overnight. The reaction was thenconcentrated in vacuo to give an orange oil of1-(2-allyloxy-4-methyl-phenyl)-3-dimethylamino-propenone (17.9 g) whichwas taken crude into the next reaction.

Step 3

To a stirred solution of crude1-(2-allyloxy-4-methyl-phenyl)-3-dimethylamino-propenone (14.1 g. 57.5mmols) in methanol (70 mL) was added hydroxylamine hydrochloride (4.4 g,63 mmols, 1.1 eq) and the reaction was stirred at room temperature for 1hour. Colourless needle crystals were filtered off and analysed andfound to be 5-(2-allyloxy-4-methyl-phenyl)-isoxazole 7.3 g (58% yield).LCMS (2 min acidic) 1.32 min 64-91% pure by UV, ES+/AP+ 216. HNMR(CDCl₃) >95% pure 7.88 (d, J=8.01 Hz, 1H), 6.88-6.92 (m, 1H), 6.82 (s,1H), 6.77 (m, 1H), 6.05-6.20 (m, 1H), 5.46 (dq, J=17.0, 1.5 Hz, 1H),5.35 (dq, J=10.5, 1.5 Hz, 1H), 4.67 (m, 2H), 2.40 (s, 3H).

Step 4

To a stirred suspension of 5-(2-allyloxy-4-methyl-phenyl)-isoxazole (7.3g, 33.8 mmols) in ethanol (40 mL) was added sodium ethoxide (21%solution in ethanol, 40 mL, 110 mmols, 3.2 eq) and the reaction wasstirred at room temperature for 3 hours. The reaction was acidified topH 2 with hydrochloric acid (2N, aqueous) and the solid was filtered offand air dried for 1 hour. The off white solid, 4.8 g (66% yield) wasanalysed and found to be pure3-(2-Allyloxy-4-methyl-phenyl)-3-oxo-propionitrile. LCMS (2 min acidic)1.12 min 51-100% pure by UV, ES+/AP+ 216, ES−/AP− 214. HNMR (CDCl₃) >95%pure 7.80 (d, J=8.0 Hz, 1H), 6.88 (dd, J=8.0, 1.0 Hz, 1H), 6.79 (s, 1H),6.13 (m, 1H), 5.37-5.50 (m, 2H), 4.67-4.70 (m, 2H), 4.08 (s, 2H), 2.41(s, 3H).

Step 5

To a stirred solution of3-(2-allyloxy-4-methyl-phenyl)-3-oxo-propionitrile (1 g, 4.6 mmols) inethanol (20 mL) was added cylohexanone (684 mg, 722 μL, 7 mmols, 1.5eq), and sulfur (224 mg, 7 mmols, 1.5 eq), followed by morpholine (607mg, 610 μL, 7 mmols, 1.5 eq) and the reaction was stirred overnight at40° C. The reaction was concentrated in vacuo. The residue was purifiedusing ISCO Companion with a Redisep silica gel 40 g cartridge and agradient of heptane and ethyl acetate (0% to 40%). Fractions containingdesired product were combined and concentrated in vacuo to give(2-allyloxy-4-methyl-phenyl)-(2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-methanoneasa yellow gum 1.1 g (72% yield). LCMS (2 min acidic) 1.45 min 58-100%pure by UV, ES+/AP+ 328. HNMR (CDCl₃) >95% pure 7.08 (d, J=7.5 Hz, 1H)6.91 (br. s., 2H) 6.78 (dq, J=7.5, 0.5 Hz, 1H) 6.69 (s, 1H) 5.92 (m,J=17.0, 10.5, 5.0, 5.0 Hz, 1H) 5.13-5.28 (m, 2H) 4.52 (dt, J=5.0, 2.0Hz, 2H) 2.47 (tt, J=6.0, 2.0 Hz, 2H) 2.36 (s, 3H) 1.78 (tt, J=6.0, 2.0Hz, 2H) 1.64-1.71 (m, 2H) 1.44-1.51 (m, 2H).

Step 6

To a stirred solution of(2-allyloxy-4-methyl-phenyl)-(2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-methanone(882 mg, 2.69 mmols) in ethanol (30 mL) was added ethyl2,4-dioxopentanoate (426 mg, 378 μL, 1 eq) followed by acetyl chloride(846 mg, 766 μL, 4 eq), and the reaction was heated to 50° C. for 1hour. The reaction was then concentrated in vacuo to give[4-(2-allyloxy-4-methyl-phenyl)-2-methyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-b]pyridin-3-yl]-oxo-aceticacid ethyl ester hydrochloride as a pale yellow oil 1 g (86% yield).LCMS (2 min acidic) 1.70 min 54-100% pure by UV, ES+/AP+ 450. HNMR(CDCl₃) >90% pure 6.95 (d, J=7.0 Hz, 1H) 6.87 (d, J=7.0 Hz, 1H) 6.76 (s,1H) 5.82 (m, 1H) 5.09-5.20 (m, 2H) 4.39-4.51 (m, 2H) 3.84-3.94 (m, 2H)2.95 (s, 3H) 2.87-2.93 (m, 2H) 2.43 (s, 3H) 1.92-2.08 (m, 2H) 1.78-1.89(m, 2H) 1.55-1.70 (m, 2H) 1.12 (t, J=7.1 Hz, 3H).

Step 7

To a stirred solution of[4-(2-Allyloxy-4-methyl-phenyl)-2-methyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-b]pyridin-3-yl]-oxo-aceticacid ethyl ester hydrochloride (1.1 g, 2.56 mmols) in ethanol (20 mL)was added sodium borohydride (145 mg, 1.5 eq, 3.84 mmols), and thereaction was stirred at room temperature for 5 minutes. The reaction wasconcentrated in vacuo, and the residue was partitioned between ethylacetate (20 mL) and hydrochloric acid (aqueous, 1 N, 30 mL). Theorganics were separated, washed with brine (10 mL), dried (MgSO4),filtered and concentrated in vacuo to give crude[4-(2-allyloxy-4-methyl-phenyl)-2-methyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-b]pyridin-3-yl]-hydroxy-aceticacid ethyl ester as a pale orange gum 1.1 g (91% yield). LCMS (2 minacidic) 1.53 min 52-81% pure by UV, ES+/AP+ 452. HNMR (CDCl₃) >80% pure7.02 (d, J=7.4 Hz, 1H) 6.81-6.85 (m, 1H) 6.79 (s, 1H) 5.86 (m, 1H) 5.18(s, 1H) 5.08-5.17 (m, 2H) 4.47-4.51 (m, 2H) 4.08-4.22 (m, 2H) 2.81 (t,J=6.2 Hz, 2H) 2.63 (s, 3H) 2.43 (s, 3H) 1.71-1.87 (m, 4H) 1.53-1.64 (m,2H) 1.17-1.21 (m, 3H).

Step 8

To a stirred solution of[4-(2-Allyloxy-4-methyl-phenyl)-2-methyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-b]pyridin-3-yl]-hydroxy-aceticacid ethyl ester (100 mg, 221 μmols, 1 eq) in MeCN (5 mL) was addedsilver oxide (102 mg, 442 μmols, 2 eq) followed by iodoethane (172 mg,89 uL, 5 eq) and the reaction was stirred for 16 hours at 60° C.Additional iodoethane (1 mL, 50 eq) and silver (I) oxide (102 mg, 2 eq)was added and the reaction was continued to be heated at 60° C. for 16hours. The reaction was diluted with MeCN (5 mL), and filtered. Thefiltrate was then concentrated in vacuo. The residue was purified usingISCO Companion with a Redisep silica gel 12 g cartridge and a gradientof heptane and ethyl acetate (0% to 30%). Fractions containing[4-(2-allyloxy-4-methyl-phenyl)-2-methyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-b]pyridin-3-yl]-ethoxy-aceticacid ethyl ester were combined and concentrated in vacuo to give thedesired product as a pale orange oil, 22 mg (22% yield). LC-MS (12 minacidic) 7.55 mins 100% pure by UV, ES+/APCI+480.

Step 9

To a stirred solution of[4-(2-Allyloxy-4-methyl-phenyl)-2-methyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-b]pyridin-3-yl]-ethoxy-aceticacid ethyl ester (22 mg, 46 μmols) in dichloromethane (3 mL) was added1,3-dimethylbarbituric acid (38 mg, 240 μmols, 5 eq), and the reactionwas evacuated and filled with nitrogen. Palladiumtetrakis(triphenylphosphine) (1.2 mg, 2 mol %) was added and thereaction was heated to reflux for 16 hours. The reaction wasconcentrated in vacuo. The residue was purified using ISCO Companionwith a Redisep silica gel 4 g cartridge and a gradient of heptane andethyl acetate (0% to 40%). Fractions containing desired product werecombined and concentrated in vacuo to giveethoxy-[4-(2-hydroxy-4-methyl-phenyl)-2-methyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-b]pyridin-3-yl]-aceticacid ethyl ester as a pale yellow oil, 20 mg (95% yield). LC-MS (12 minacidic) 6.40 mins 58-69% pure by UV, ES+/APCI+440, ES−/APCI− 438; 6.66mins 27-31% pure by UV, ES+/APCI+440, ES−/APCI− 438.

Step 10

To a stirred solution ofEthoxy-[4-(2-hydroxy-4-methyl-phenyl)-2-methyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-b]pyridin-3-yl]-aceticacid ethyl ester (20 mg, 45 μmols) in ethanol (1 mL) and tetrahydrofuran(1 mL) was added the sodium hydroxide (2N aqueous, 0.5 mL, 20 eq) andthe reaction was stirred at 60° C. for 5 hours. The reaction wasconcentrated in vacuo until all organic solvents had been removed, theaqueous residue was then acidified with hydrochloric acid (2N aqueous)to pH 2. A pale yellow solid, 4 mg (16% yield) was filtered off andanalysed and found to containethoxy-[4-(2-hydroxy-4-methyl-phenyl)-2-methyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-b]pyridin-3-yl]-aceticacid. LC-MS (12 min acidic) 5.04 mins 47-43% pure by UV, ES+/APCI+412,ES−/APCI− 410; 5.80 mins 12-13% pure by UV, ES+/APCI+412, ES−/APCI− 410.

EXAMPLE 42-tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetic acid

5-Benzothiazole-boronic acid (150 μmol) andethyl-2-tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetate(Preparation 11, 1 mL of a 0.1M solution in dioxane, 100 μmol) wereadded to a reaction vial. A 1M solution of caesium carbonate (2004, 200μmol) in water was then added, followed by 5 μmol of Pd(dppf)Cl₂ and thewhole stirred at 100° C. for 16h before cooling to room temperature andevaporation of the solution under reduced pressure to give a yellowresidue. A 2M solution of lithium hydroxide in water (2004, 400 μmol)was added to each vial, followed by 1 mL of THF, and the mixture wasshaken at room temperature for 16h. The pH of the solution was adjustedto neutral using a 1M aqueous solution of hydrochloric acid, before themixture was evaporated to dryness under reduced pressure and the residuethen purified by preparative HPLC on a C18 column, using a mixture ofacetonitrile and water as the mobile phase. Evaporation of theappropriate fractions under reduced pressure provided the title compound(11 mg, 23%) as a white solid. ESI/APC(+): 467 (M+H).

The following Examples were synthesised according to the methoddescribed in Example 4 using the appropriate aryl boronic acid.

Antiviral Activity HTRF Interaction Example Mass Structure (S8737E, μM)assay (S9118, μM)  4 467

0.282 (n = 2) 3.48 (n = 2)  5 454

0.155 (n = 2) 2.7 (n = 2)  6 450

0.305 (n = 2) 2.63 (n = 2)  7 441

0.436 (n = 2) 9.25 (n = 2)  8 428

0.624 (n = 2) 23.8 (n = 2)  9 479

0.683 (n = 2) 11.7 (n = 2) 10 464

0.795 (n = 2) 7.87 (n = 2) 11 464

0.814 (n = 2) 12.2 (n = 2) 12 464

0.941 (n = 2) 5.13 (n = 2) 13 440

0.957 (n = 2) 56.3 (n = 2) 14 441

1.24 (n = 2) 28.4 (n = 2) 15 435

1.28 (n = 2) 14.8 (n = 2) 16 426

1.73 (n = 2) 57.4 (n = 2) 17 450

1.78 (n = 2) >100 (n = 2) 18 451

1.84 (n = 2) 30.3 (n = 2) 19 490

1.9 (n = 2) 46.5 (n = 2) 20 481

2.01 (n = 2) 11.8 (n = 2) 21 440

2.17 (n = 2) 64.8 (n = 2) 22 481

2.73 (n = 2) 12.8 (n = 2) 23 422

2.73 (n = 2) 33.1 (n = 2) 24 464

2.81 (n = 2) 7.59 (n = 2) 25 397

4.07 (n = 2) >100 (n = 2) 26 435

4.51 (n = 2) >100 (n = 2) 27 440

4.64 (n = 2) 57.5 (n = 2) 28 504

5.61 (n = 2) 29.6 (n = 2) 29 454

5.73 (n = 2) 64.6 (n = 2) 30 450

6.63 (n = 2) 15.5 (n = 2) 31 439

6.66 (n = 2) >100 (n = 2) 32 450

7.65 (n = 2) >100 (n = 2) 33 450

8.19 (n = 2) >100 (n = 2) 34 412

8.97 (n = 2) >100 (n = 2) 35 436

10.7 (n = 2) >100 (n = 2) 36 450

12 (n = 2) >100 (n = 2) 37 425

13.9 (n = 2) >100 (n = 2) 38 476

15.1 (n = 2) 97.5 (n = 2) 39 442

15.5 (n = 2) >100 (n = 2) 40 436

15.7 (n = 2) >100 (n = 2) 41 400

17.7 (n = 2) >100 (n = 2) 42 413

18.1 (n = 2) >100 (n = 2) 43 436

18.9 (n = 2) >100 (n = 2)

EXAMPLE 44(2S)-tert-butoxy[4-(4-chlorophenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid

Step 1: Methyl(2S)-tert-butoxy[4-(4-chlorophenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]acetate

To a solution of Methyl(2S)-tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetate(Preparation 16, 100 mg, 212 μmol) in dioxane (2 mL) was added4-chlorobenzene boronic acid (66 mg, 422 μmol), ethyl-di-isopropyl-amine(120 μL, 636 μmol), water (500 μL) and palladiumtetrakis(triphenylphosphine) (25 mg, 20 μmol). The reaction mixture wasdegassed and stirred at 100° C. in a sealed tube. The reaction wascooled to room temperature and diluted with ethyl acetate (10 mL). Themixture was passed through a pad of Celite. The organic filtrate waswashed with water (10 mL) and brine (10 mL), dried over MgSO₄ andconcentrated in vacuo to yield the crude product. The residue waspurified by flash chromatography eluting with ethyl acetate in heptane(0-6%) to give the title compound as a white semi-solid, 60 mg, in a 62%yield. ¹H NMR (400 MHz, CDCl₃) δ ppm 0.97 (s, 9H), 1.45-1.48 (m, 1H),1.62-1.73 (m, 3H), 1.78-1.82 (m, 2H), 2.71 (s, 3H), 2.78-2.81 (m, 2H),3.66 (s, 3H), 4.99 (s, 1H), 7.18-7.21 (m, 1H), 7.39-7.42 (m, 3H).

Step 2:(2S)-tert-butoxy[4-(4-chlorophenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid

To a solution of Methyl(2S)-tert-butoxy[4-(4-chlorophenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]acetate(60 mg, 131 μmol) in tetrahydrofuran (2 mL) and methylated spirit (2 mL)was added an aqueous sodium hydroxide solution (1 M, 790 μL, 790 μmol).The resulting solution was stirred at 60° C. for 3 hours. The volatilesolvents were removed in vacuo and the residue diluted with water (10mL). Dichloromethane (20 mL) was added to the mixture, which was thenacidified to pH 5 by the addition of 2 M aqueous hydrochloric acid. Theorganic layer was separated and washed with water (10 mL) and brine (10mL), dried over MgSO₄ and concentrated in vacuo. The residue wasrecrystallised from 2-propanol and the resulting solid collected byfiltration to give the title compound as a white solid, 11.2 mg, in a19% yield.

¹H NMR (400 MHz, CDCl₃) δ ppm 1.02 (s, 9H), 1.45-1.48 (m, 1H), 1.67-1.73(m, 3H), 1.78-1.84 (m, 2H), 2.69 (s, 3H), 2.79-2.81 (m, 2H), 5.11 (s,1H), 7.18-7.22 (m, 1H), 7.42-7.46 (m, 2H), 7.59-7.61 (m, 1H).

Antiviral Activity=0.036 μM (n=6) (S8737E).

HTRF Interaction assay=576 nM (n=10) (S9118)

EXAMPLE 45(2S)-tert-butoxy[4-(4-chloro-2-fluorophenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid

Step 1: Methyl(2S)-tert-butoxy[4-(4-chloro-2-fluorophenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]acetate

The title compound was prepared from Methyl(2S)-tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetate(Preparation 16, 100 mg, 212 μmol) and (4-chloro-2-fluorophenyl) boronicacid (74 mg, 424 μmol) using the same method as described in Example 44,Step 1 to yield 57 mg, 56%. Material obtained was a mixture ofatropisomers, in a 3:1 ratio. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.01 (s,9H), 1.48-1.55 (m, 1H), 1.69-1.75 (m, 3H), 1.95-2.03 (m, 1H), 2.72 (s,3H), 2.80-2.86 (m, 3H), 3.66 (s, 3H), 4.98 (s, 1H), 7.19-7.21 (m, 1H),7.25-7.27 (m, 1H), 7.35-7.37 (m, 1H).

Step 2:(2S)-tert-butoxy[4-(4-chloro-2-fluorophenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid

The title compound was prepared from Methyl(2S)-tert-butoxy[4-(4-chloro-2-fluorophenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]acetate(57 mg, 119 μmol) using the same method as described in Example 44, Step2. Purification by flash column chromatography, eluting with 5% methanolin dichloromethane gave the title compound, 24 mg, in a 24% yield.Material is a single atropisomer. The second atropisomer was notisolated. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.04 (s, 9H), 1.45-1.51 (m, 1H),1.62-1.76 (m, 3H), 1.82-1.84 (m, 1H), 2.05-2.10 (m, 1H), 2.69 (s, 3H),2.80-2.84 (m, 2H), 5.08 (s, 1H), 7.22-7.28 (m, 2H), 7.59-7.61 (m, 1H).

Antiviral Activity=0.023 μM (n=6) (S8737E).

HTRF Interaction assay=565 nM (n=10) (S9118)

EXAMPLE 46(2S)-tert-butoxy[4-(4-chloro-2-hydroxyphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid

Step 1: Methyl(2S)-tert-butoxy[4-(4-chloro-2-hydroxyphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]acetate

To a solution of Methyl(2S)-tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetate(Preparation 16, 200 mg, 422 μmol) in dioxane (4 mL) was added(4-chloro-2-hydroxyphenyl) boronic acid (Preparation 17, 191 mg, 761μmol), Dichloro [1,1′ bis(di-tert-butylphosphino)]ferrocene palladium(II)™ (Pd-118) (Johnson-Matthey, 27 mg, 10 mol %, 42.2 μmol), potassiumphosphate (180 mg, 844 μmol) and water (1 mL). The resulting mixture wasstirred at 105° C. in a sealed tube for 18 hours. A further portion ofPd-118 (10 mg, 3.7 mol %, 15.6 μmol) was added and the mixture stirredat 105° C. for 72 hours. The reaction mixture was cooled to roomtemperature and filtered through a short pad of silica, washing withethyl acetate. The solvent was removed in vacuo. The crude product waspurified by column chromatography eluting with ethyl acetate/heptane(0-10%) to yield the title compound as a brown solid, 37.5 mg, in a 19%yield. Material obtained was a mixture of atropisomers, in a ˜2:1 ratioand was carried on to hydrolysis step 2 without further purification.

Step 2:(2S)-tert-butoxy[4-(4-chloro-2-hydroxyphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid

The title compound was prepared from Methyl(2S)-tert-butoxy[4-(4-chloro-2-hydroxyphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]acetate(70 mg, 148 μmol) using the same method as described in Example 44, Step2. Purification by flash column chromatography, eluting with ethylacetate, then with 20% methanol in ethyl acetate gave the separatedatropisomers, 46A: 26 mg, in a 24% yield and 46B: 7 mg, in a 10% yield.46A: ¹H NMR (400 MHz, CD₃OD) δ ppm 1.02 (s, 9H), 1.50-1.53 (m, 1H),1.65-1.98 (m, 4H), 2.18-2.21 (m, 1H), 2.64 (s, 3H), 2.79-2.81 (m, 2H),5.13 (s, 1H), 6.91 (s, 1H), 6.97 (d, 1H), 7.27 (d, 1H). 46B: ¹H NMR (400MHz, CD₃OD) δ ppm 1.00 (s, 9H), 1.49-1.51 (m, 1H), 1.65-1.85 (m, 4H),2.20-2.26 (m, 1H), 2.69 (s, 3H), 2.79-2.81 (m, 2H), 5.05 (s, 1H), 6.88(s, 1H), 6.92 (d, 1H), 7.40 (d, 1H).

Atropisomer 46A: Antiviral activity=0.014 μM (n=2) (S8737E)

-   -   HTRF Interaction assay=558 nM (n=6) (S9118)

Atropisomer 46B: Antiviral Activity=0.040 μM (n=2) (S8737E).

-   -   HTRF Interaction assay=798 nM (n=2) (S9118)

EXAMPLE 47(2S)-tert-butoxy[4-(2-fluoro-4-methylphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid

Step 1: Methyl(2S)-tert-butoxy[4-(2-fluoro-4-methylphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]acetate

The title compound was prepared from Methyl(2S)-tert-butoxy(4-iodo-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl)acetate(Preparation 16, 100 mg, 212 μmol) and2-(2-fluoro-4-methylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(Preparation 19, 99 mg, 424 μmol) using the same method as described inExample 44, Step 1 to yield 100 mg, 96%. Material obtained was a mixtureof atropisomers, in a 2:1 ratio.

¹H NMR (400 MHz, CDCl₃) δ ppm 0.99 (s, 5.8H), 1.05 (s, 3.2H), 1.26-2.08(m, 6H), 2.45 (s, 3H), 2.71 (s, 1.8H), 2.76-2.80 (m, 2H), 2.81 (s,1.2H), 3.58 (s, 1.2H), 3.66 (s, 1.8H), 5.06 (s, 0.66H), 5.07 (s, 0.33H),6.94-7.02 (m, 2.4H), 7.23-7.25 (m, 0.6H).

Step 2:(2S)-tert-butoxy[4-(2-fluoro-4-methylphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid

The title compound was prepared from Methyl(2S)-tert-butoxy[4-(2-fluoro-4-methylphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]acetate(100 mg, 220 μmol) using the same method as described in Example 44,Step 2. Purification by flash column chromatography, eluting with 5%methanol in dichloromethane failed to separate the atropisomers, and themixture was still a 2:1 ratio of two atropisomers, 40 mg, 41%. ¹H NMR(400 MHz, CDCl₃) δ ppm 0.95 (s, 5.8H), 1.05 (s, 3.2H), 1.26-2.08 (m,6H), 2.44 (s, 1.2H), 2.45 (s, 1.8H), 2.68 (s, 1.8H), 2.75 (s, 1.2H),2.76-2.81 (m, 2H), 5.05 (s, 0.66H), 5.11 (s, 0.33H), 7.02-7.13 (m,2.4H), 7.45-7.49 (m, 0.6H).

Antiviral Activity=0.071 μM (n=2) (S8737E).

HTRF Interaction assay=657 nM (n=2) (S9118)

EXAMPLE 48Tert-butoxy[4-(2-hydroxy-4-methylphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid

Step 1: 1-[2-(allyloxy)-4-methylphenyl]ethanone

To a solution of 1-(2-hydroxy-4-methyl-phenyl)-ethanone (10 g, 67 mmol)in dimethylformamide (100 mL) was added potassium carbonate (18.4 g, 133mmol), followed by allyl bromide (5.75 mL, 67 mmol). The reaction wasstirred at room temperature for 16 hours. The residue was partitionedbetween ethyl acetate (200 mL) and water (800 mL), the organics wereseparated, and washed with brine (50 mL), dried over MgSO₄ andconcentrated in vacuo. After cooling the concentrate to room temperaturethe title compound crystallised as colourless platelets, 12.40 g, in a98% yield. LCMS (2 min acidic) 1.20 min 72-100% pure by UV, ES+/AP+191.¹HNMR (400 MHz, CDCl₃) δ ppm 2.37 (s, 3H), 2.63 (s, 3H), 4.61-4.66 (m,2H), 5.33 (dq, 1H), 5.44 (dq, 1H), 6.10 (m, 1H), 6.76 (s, 1H), 6.80-6.83(m, 1H), 7.68 (d, 1H).

Step 2:(2E)-1-[2-(allyloxy)-4-methylphenyl]-3-(dimethylamino)prop-2-en-1-one

To 1-[2-(allyloxy)-4-methylphenyl]ethanone (Step 1, 13.9 g, 73 mmol) wasadded dimethylformamide dimethylacetal (56 mL, 422 mmol), and thereaction was heated to reflux for 18 hours. The reaction was thenconcentrated in vacuo to give the title compound as an orange oil, 17.9g, which was taken crude into the next reaction.

Step 3: 5-[2-(allyloxy)-4-methylphenyl]isoxazole

To a stirred solution of crude(2E)-1-[2-(allyloxy)-4-methylphenyl]-3-(dimethylamino)prop-2-en-1-one(Step 2, 14.1 g, 57.5 mmol) in methanol (70 mL) was added hydroxylaminehydrochloride (4.4 g, 63 mmol) and the reaction was stirred at roomtemperature for 1 hour. Colourless needle crystals were filtered off andanalysed and found to be the title compound, 7.3 g, in a 58% yield. LCMS(2 min acidic) 1.32 min 64-91% pure by UV, ES+/AP+ 216. ¹HNMR (400 MHz,CDCl₃) δ ppm 2.40 (s, 3H), 4.67 (m, 2H), 5.35 (dq, 1H), 5.46 (dq, 1H),6.05-6.20 (m, 1H), 6.77 (m, 1H), 6.82 (s, 1H), 6.88-6.92 (m, 1H), 7.88(d, 1H).

Step 4: 3-[2-(allyloxy)-4-methylphenyl]-3-oxopropanenitrile

To a stirred suspension of 5-[2-(allyloxy)-4-methylphenyl]isoxazole(Step 3, 7.3 g, 33.8 mmol) in ethanol (40 mL) was added sodium ethoxide(21% solution in ethanol, 40 mL, 110 mmol) and the reaction was stirredat room temperature for 3 hours. The reaction was acidified to pH 2 withhydrochloric acid (2N, aqueous) and the solid was filtered off and airdried for 1 hour to give the title compound as an off-white solid, 4.8g, in a 66% yield. LCMS (2 min acidic) 1.12 min 51-100% pure by UV,ES+/AP+ 216, ES−/AP− 214. ¹HNMR (400 MHz, CDCl₃) δ ppm 2.41 (s, 3H),4.08 (s, 2H), 4.67-4.70 (m, 2H), 5.37-5.50 (m, 2H), 6.13 (m, 1H), 6.79(s, 1H), 6.88 (dd, 1H), 7.80 (d, 1H).

Step 5:[2-(allyloxy)-4-methylphenyl](2-amino-4,5,6,7-tetrahydro-1-benzothien-3-yl)methanone

To a stirred solution of3-[2-(allyloxy)-4-methylphenyl]-3-oxopropanenitrile (Step 4, 1 g, 4.6mmol) in ethanol (20 mL) was added cyclohexanone (722 μL, 7 mmol) andsulfur (224 mg, 7 mmol), followed by morpholine (610 μL, 7 mmol) and thereaction was stirred overnight at 40° C. The reaction was concentratedin vacuo. The residue was purified using ISCO Companion with a Redisepsilica gel 40 g cartridge and a gradient of heptane and ethyl acetate(0% to 40%). Fractions containing desired product were combined andconcentrated in vacuo to give the title compound as a yellow gum, 1.1 g,in a 72% yield. LCMS (2 min acidic) 1.45 min 58-100% pure by UV, ES+/AP+328. ¹HNMR (400 MHz, CDCl₃) δ ppm 1.44-1.51 (m, 2H), 1.64-1.71 (m, 2H),1.78 (tt, 2H), 2.36 (s, 3H), 2.47 (tt, 2H), 4.52 (dt, 2H), 5.13-5.28 (m,2H), 5.92 (m, 1H), 6.69 (s, 1H), 6.78 (dq, 1H), 6.91 (br s, 2H), 7.08(d, 1H).

Step 6: Ethyl{4-[2-(allyloxy)-4-methylphenyl]-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl}(oxo)acetatehydrochloride salt

To a stirred solution of[2-(allyloxy)-4-methylphenyl](2-amino-4,5,6,7-tetrahydro-1-benzothien-3-yl)methanone(Step 5, 882 mg, 2.69 mmol) in ethanol (30 mL) was added ethyl2,4-dioxopentanoate (378 μL, 2.69 mmol) followed by acetyl chloride (766μL, 10.8 mmol), and the reaction was heated to 50° C. for 1 hour. Thereaction was then concentrated in vacuo to give a mixture ofdiastereoisomers of the title compound as the hydrochloride salt, as apale yellow oil, 1 g, in an 86% yield. LCMS (2 min acidic) 1.70 min54-100% pure by UV, ES+/AP+ 450. ¹HNMR (400 MHz, CDCl₃) δ ppm 1.12 (t,3H), 1.55-1.70 (m, 2H), 1.78-1.89 (m, 2H), 1.92-2.08 (m, 2H), 2.43 (s,3H), 2.87-2.93 (m, 2H), 2.95 (s, 3H), 3.84-3.94 (m, 2H), 4.39-4.51 (m,2H), 5.09-5.20 (m, 2H), 5.82 (m, 1H), 6.76 (s, 1H), 6.87 (d, 1H), 6.95(d, 1H).

Step 7: Ethyl{4-[2-(allyloxy)-4-methylphenyl]-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl}(hydroxy)acetate

To a stirred solution of ethyl{4-[2-(allyloxy)-4-methylphenyl]-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl}(oxo)acetatehydrochloride salt (Step 6, 1.1 g, 2.56 mmol) in ethanol (20 mL) wasadded sodium borohydride (145 mg, 3.84 mmol), and the reaction wasstirred at room temperature for 5 minutes. The reaction was concentratedin vacuo, and the residue was partitioned between ethyl acetate (20 mL)and hydrochloric acid (aqueous, 1 N, 30 mL). The organic layer wasseparated, washed with brine (10 mL), dried over MgSO₄, filtered andconcentrated in vacuo to give a mixture of diastereoisomers of the titlecompound as a pale orange gum, 1.1 g, in a 91% yield. LCMS (2 minacidic) 1.53 min 52-81% pure by UV, ES+/AP+452. ¹HNMR (400 MHz, CDCl₃ δppm 1.17-1.21 (m, 3H), 1.53-1.64 (m, 2H), 1.71-1.87 (m, 4H), 2.43 (s,3H), 2.63 (s, 3H), 2.81 (t, 2H), 4.08-4.22 (m, 2H), 4.47-4.51 (m, 2H),5.08-5.17 (m, 2H), 5.18 (s, 1H), 5.86 (m, 1H), 6.79 (s, 1H), 6.81-6.85(m, 1H), 7.02 (d, 1H).

Step 8: Ethyl{4-[2-(allyloxy)-4-methylphenyl]-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl}(tert-butoxy)acetate

To a stirred solution of ethyl{4-[2-(allyloxy)-4-methylphenyl]-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl}(hydroxy)acetate(Step 7, 720 mg, 1.59 mmol) in dichloromethane (2.5 mL) was addedtert-butyl acetate (2.5 mL) followed by concentrated sulphuric acid (244μL, 4.78 mmol) and the reaction was stirred at room temperature for 2hours. The reaction mixture was quenched by addition of a 1 M aqueoussodium hydroxide solution until the solution was at pH 5. The volatilesolvents were removed in vacuo, and the remaining aqueous layer wasextracted with ethyl acetate (30 mL). The organic layer was washed withbrine (10 mL), dried over MgSO₄ and concentrated in vacuo. The residuewas purified using ISCO Companion with a Redisep silica gel 12 gcartridge and a gradient of heptane and ethyl acetate (0% to 40%).Product containing fractions were concentrated in vacuo to give amixture of diastereoisomers of the title compound as a colourless oil,370 mg, in a 45% yield. Material is shown to be a mixture ofdiastereomers, in an approximately 80:20 ratio.

Major diastereomer (˜80%)

¹H NMR (400 MHz, CDCl₃) δ ppm 0.95 (s, 9H) 1.19 (t, 3H) 1.53-1.82 (m,4H) 1.89-1.99 (m, 2H) 2.42 (s, 3H) 2.70 (s, 3H) 2.80 (m, 2H) 4.11 (m,2H) 4.26-4.45 (m, 2H) 4.94-5.04 (m, 2H) 5.05 (s, 1H) 5.70-5.80 (m, 1H)6.71 (s, 1H) 6.80 (d, 1H) 7.15 (d, 1H).

Minor diastereomer (˜20%)

¹H NMR (400 MHz, CDCl₃) δ ppm 1.00 (s, 9H) 1.13 (t, 3H) 1.53-1.82 (m,4H) 1.89-1.99 (m, 2H) 2.42 (s, 3H) 2.70 (s, 3H) 2.80 (m, 2H) 4.11 (m,2H) 4.26-4.45 (m, 2H) 4.94-5.04 (m, 2H) 5.05 (s, 1H) 5.70-5.80 (m, 1H)6.73 (s, 1H) 6.78 (d, 1H) 6.93 (d, 1H).

Step 9: Ethyltert-butoxy[4-(2-hydroxy-4-methylphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]acetate

To a stirred solution of ethyl{4-[2-(allyloxy)-4-methylphenyl]-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl}(tert-butoxy)acetate(Step 8, 370 mg, 729 μmol) in dichloromethane (10 mL) was added1,3-dimethylbarbituric acid (569 mg, 3.64 mmol), and the reaction wasevacuated and filled with nitrogen. Palladiumtetrakis(triphenylphosphine) (17 mg, 15 μmol) was added and the reactionwas heated to reflux for 16 hours. Further palladiumtetrakis(triphenylphosphine) (17 mg, 15 μmol) was added and the reactionwas heated to reflux for a further 4 hours. The reaction wasconcentrated in vacuo and preabsorbed onto silica. The residue waspurified using ISCO Companion with a Redisep silica gel 12 g cartridgeand a gradient of heptane and ethyl acetate (0% to 20%). Productcontaining fractions were concentrated in vacuo to give the titlecompound as a pair of diastereoisomers as a colourless oil, 214 mg, in a63% yield. The other pair of minor diastereoisomers co-eluted withunreacted starting material and were not isolated. LC-MS (12 min acidic)6.89 mins 100% pure by UV, ES+/APCI+ 468, ES−/APCI− 466 ¹H NMR (400 MHz,CDCl₃) δ ppm 1.01 (s, 9H) 1.20 (t, 3H) 1.62-1.85 (m, 4H) 2.02-2.14 (m,2H) 2.41 (s, 3H) 2.74 (s, 3H) 2.78-2.85 (m, 2H) 4.08-4.18 (m, 2H) 5.14(s, 1H) 6.78 (s, 1H) 6.82 (d, 1H) 7.17 (d, 1H).

Step 10:Tert-butoxy[4-(2-hydroxy-4-methylphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid

To a stirred solution of the major diastereoisomeric pair of ethyltert-butoxy[4-(2-hydroxy-4-methylphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]acetate(Step 9, 214 mg, 458 μmol) in ethanol (5 mL) and tetrahydrofuran (5 mL)was added a solution of sodium hydroxide (2 N, aqueous, 2 mL, 2.75 mmol)and the reaction was stirred at 60° C. for 18 hours. The reaction wasconcentrated in vacuo until all organic solvents had been removed, theaqueous residue was then acidified with hydrochloric acid (2 N, aqueous)to pH 2. The precipitated solid was collected by filtration and washedwith tert-butyl methyl ether (10 mL) and dried in vacuo to give amixture of diastereoisomers of the title compound as a white solid, 76mg, in a 38% yield. LC-MS (12 min acidic) 5.51 mins 81% pure by UV,ES+/APCI+440, ES−/APCI− 438. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.04 (s, 9H)1.66-1.88 (m, 4H) 2.09-2.21 (m, 2H) 2.40 (s, 3H) 2.73 (s, 3H) 2.82 (t,2H) 5.33 (s, 1H) 6.78 (s, 1H) 6.85 (d, 1H) 7.36 (d, 1H).

Step 11: Chiral Separation

The diastereomeric pair isolated in Step 10 was separated using aChiralpak IC column, eluting with 70:30 Heptane:IPA at 18 mL per minute,and a total run time of 9 minutes. The first enantiomer eluted at 3.56minutes and the second enantiomer eluted at 4.54 minutes, monitoring byUV.

The first eluting enantiomer was confirmed as(2R)-tert-butoxy[4-(2-hydroxy-4-methylphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid.

Antiviral Activity=0.089 μM (n=2) (S8737E).

HTRF Interaction assay=4120 nM (n=2) (S9118)

The second eluting enantiomer was confirmed as(2S)-tert-butoxy[4-(2-hydroxy-4-methylphenyl)-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-b]pyridin-3-yl]aceticacid with the atropisomeric configuration shown below (confirmed byX-ray structure):

Antiviral Activity=0.013 μM (n=2) (S8737E).

HTRF Interaction assay=576 nM (n=2) (S9118)

EXAMPLE 49 Preparation of(S)-2-(tert-butoxy)-2-(4-(4-(difluoromethyl)phenyl)-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)aceticacid

Step 1: (S)-methyl2-(tert-butoxy)-2-(4-(4-(difluoromethyl)phenyl)-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)acetate

2-(4-(Difluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1.04 g, 4.09 mmol), potassium phosphate (1.35 g, 6.36 mmol), water (750μL) and dichloro [1,1′ bis(di-tert-butylphosphino)]ferrocene palladium(II)™ (101 mg, 155 μmol) were added to a stirred solution of (S)-methyl2-(tert-butoxy)-2-(4-iodo-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)acetate(750 mg, 1.58 mmol) in dioxane (11 mL) in a reaction tube. The reactionmixture was degassed with argon for 2 minutes, sealed and then stirredat 100° C. for 16 hours. The reaction mixture was cooled to roomtemperature, diluted with ethyl acetate (30 mL) and water (30 mL) andthen passed through a pad of celite. The layers of the filtrate wereseparated and the aqueous layer was extracted with ethyl acetate (3×30mL). The combined organic layers were dried (Na₂SO₄) and concentrated invacuo to yield the crude product. The residue was purified by flashcolumn chromatography eluting with ethyl acetate in heptane (10%) togive the title compound (639 mg, 85%) as a yellow oil. ¹H NMR (400 MHz,CDCl₃) δ=0.96 (s, 9H), 1.85-1.35 (m, 6H), 2.72 (s, 3H), 2.85-2.75 (m,2H), 3.67 (s, 3H), 4.95 (s, 1H), 6.76 (t, 1H), 6.45 (d, 1H), 7.61-7.51(m, 3H). LCMS (run time=5 minutes, basic): R_(t)=3.37 minutes; m/z474.23 [M+H⁺].

Step 2:(S)-2-(tert-butoxy)-2-(4-(4-(difluoromethyl)phenyl)-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)aceticacid

2 M Aqueous sodium hydroxide solution (1.93 mL, 3.86 mmol) was added toa solution of (S)-methyl2-(tert-butoxy)-2-(4-(4-(difluoromethyl)phenyl)-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)acetate (183 mg, 0.39 mmol) in 1:1tetrohydrofuran/industrial methylated spirit (4 mL) at room temperature.The resulting mixture stirred at room temperature for 64 h. The reactionmixture was partitioned between ethyl acetate (20 mL) and water (10 mL).The aqueous solution was adjusted to pH 4 by the addition of 2 M aqueoushydrochloric acid solution. The layers were separated and the aqueouslayer was extracted with ethyl acetate (2×10 mL). The combined organiclayers were dried (MgSO₄) and concentrated in vacuo to give the crudeproduct as a pale orange solid. This was purified by flash columnchromatography on silica eluting with methanol in dichloromethane (3-5%)to give the title compound (111 mg, 63%) as an off-white solid. ¹H NMR(400 MHz, CDCl₃) δ=1.01 (s, 9H), 1.90-1.35 (m, 6H), 2.70 (s, 3H),2.85-2.78 (m, 2H), 5.07 (brs, 1H), 6.75 (t, 1H), 7.34-7.41 (brm, 1H),7.61 (d, 2H), 7.80-7.70 (brm, 1H). LCMS (run time=5 minutes, basic):R_(t)=3.04 minutes; m/z 460.22 [M+H⁺].

Antiviral Activity=0.081 μM (n=6) (S8737E).

HTRF Interaction assay=11010 nM (n=6) (S9118)

EXAMPLE 50 Preparation of(S)-2-(tert-butoxy)-2-(2-methyl-4-(4-(trifluoromethyl)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)aceticacid

Step 1: (S)-methyl2-(tert-butoxy)-2-(2-methyl-4-(4-(trifluoromethyl)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)acetate

2-(4-(Trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(115 g, 0.42 mmol), N-ethyl-N-isopropylpropan-2-amine (120 μL, 0.64mmol) and water (500 μL) were added to a stirred solution of (S)-methyl2-(tert-butoxy)-2-(4-iodo-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)acetate(100 mg, 0.21 mmol) in dioxane (2 mL) in a reaction tube. The reactionmixture was degassed with argon for 2 minutes, thentetrakis(triphenylphosphine)palladium(0) (25 mg, 21 μmmol) was added andthe vessel was sealed and heated at 100° C. for 16 hours. The reactionmixture was cooled to room temperature and diluted with ethyl acetate(30 mL) and water (30 mL) and the mixture was passed through a pad ofcelite. The layers of the filtrate were separated and the organic layerwas washed with brine (30 mL), dried (MgSO₄) and concentrated in vacuoto yield the crude product as a brown gum. The residue was purified byflash column chromatography eluting with ethyl acetate in heptane (10%)to give the title compound (75 mg, 72%) as a yellow oil. ¹H NMR (400MHz, CDCl₃) δ=0.96 (s, 9H), 1.85-1.35 (m, 6H), 2.72 (s, 3H), 2.85-2.75(m, 2H), 3.69 (s, 3H), 4.91 (s, 1H), 7.40 (d, 1H), 7.61 (d, 1H), 7.71(m, 2H). LCMS (run time=5 minutes, basic): R_(t)=3.64 minutes; m/z492.06 [M+H⁺].

Step 2:(S)-2-(tert-butoxy)-2-(2-methyl-4-(4-(trifluoromethyl)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)aceticacid

1 M Aqueous sodium hydroxide solution (1.6 mL, 1.6 mmol) was added to asolution of (S)-methyl2-(tert-butoxy)-2-(2-methyl-4-(4-(trifluoromethyl)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)acetate (75 mg, 0.15 mmol) in 1:1tetrhydrofuran/industrial methylated spirit (4 mL) at room temperature.The resulting mixture was heated at 60° C. 1.5 h. The resulting solutionwas concentrated in vacuo and the residue was dissolved in water (10mL). The aqueous solution was adjusted to pH 4 by the addition of 2 Maqueous hydrochloric acid solution and the resulting suspension wasextracted with dichloromethane (20 mL). The organic layer was washedwith brine, dried (MgSO₄) and concentrated in vacuo to give the crudeproduct as a pale yellow solid. This was purified by flash columnchromatography on silica eluting with methanol in dichloromethane (5%)to give the title compound (40.3 mg, 56%) as a pale yellow solid. ¹H NMR(400 MHz, CDCl₃) δ=1.01 (s, 9H), 1.89-1.30 (m, 6H), 2.71 (s, 3H),2.90-2.75 (m, 2H), 5.02 (s, 1H), 7.48-7.34 (m, 1H), 7.90-7.64 (m, 3H).LCMS (run time=5 minutes, basic): R_(t)=3.30 minutes; m/z 478.01 [M+H⁺].

Antiviral Activity=0.055 μM (n=4) (S8737E).

HTRF Interaction assay=1500 nM (n=4) (S9118)

EXAMPLE 51 Preparation of(S)-2-(4-(4-bromophenyl)-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)-2-(tert-butoxy)aceticacid

Step 1: (S)-methyl2-(4-(4-aminophenyl)-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)-2-(tert-butoxy)acetate

4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (140 mg, 0.63mmol), sodium hydrogen carbonate (178 mg, 2.1 mmol) and water (200 μL)were added to a stirred solution of (S)-methyl2-(tert-butoxy)-2-(4-iodo-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)acetate (200 mg, 0.42 mmol) in N,N-dimethylacetamide (4 mL) in a reaction tube. The reaction mixture wasdegassed with argon for 2 minutes, thenbis(tri-tert-butylphosphine)palladium(0) (22 mg, 42 μmmol) was added andthe vessel was sealed and heated at 100° C. for 16 hours. The reactionmixture was cooled to room temperature and partitioned betweendichloromethane (30 mL) and water (30 mL). The layers were separated andthe organic layer was dried (MgSO₄) and concentrated in vacuo to yieldthe crude product as a brown gum. The residue was purified by flashcolumn chromatography on silica eluting with ethyl acetate in heptane(25%) to give the title compound (114 mg, 67%) as a white foam. ¹H NMR(400 MHz, CDCl₃) δ=0.97 (s, 9H), 1.95-1.30 (m, 6H), 2.68 (s, 3H),2.85-2.77 (m, 2H), 3.65 (s, 3H), 3.80 (brs, 2H), 5.19 (s, 1H), 6.75-6.68(m, 2H), 6.99 (d, 1H), 7.19 (d, 1H).

Step 2: (S)-methyl2-(4-(4-bromophenyl)-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)-2-(tert-butoxy)acetate

A solution of copper(II)bromide (73 mg, 0.33 mmol) and tert-butylnitrite(60 μL, 0.43 mmol) in acetonitrile (2 mL) was added to a stirredsolution of (S)-methyl2-(4-(4-aminophenyl)-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)-2-(tert-butoxy)acetate(114 mg, 0.26 mmol) in acetonitrile (1 mL) at room temperature and theresulting mixture stirred at room temperature for 16 hours. The mixturewas partitioned between 2 M aqueous hydrochloric acid solution (5 mL)and dichloromethane (10 mL) were added. The layers were separated andthe organic layer was washed with water (10 mL), brine (10 mL), dried(MgSO₄) and concentrated in vacuo to give the crude product. This waspurified by flash column chromatography on silica eluting with ethylacetate in hepatane (10%) to give the title compound (83 mg, 63%) asbrown oil. ¹H NMR (400 MHz, CDCl₃) δ=0.96 (s, 9H), 1.85-1.40 (m, 6H),2.71 (S, 3H), 2.81-2.75 (m, 2H), 3.65 (s, 3H), 4.99 (s, 1H), 7.12 (s,1H), 7.31 (d, 1H), 7.60-7.50 (m, 2H). LCMS (run time=5 minutes, basic):R_(t)=3.57 minutes; m/z 504.12 [M+H⁺].

Step 3:(S)-2-(4-(4-bromophenyl)-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)-2-(tert-butoxy)aceticacid

1 M Aqueous sodium hydroxide solution (1.6 mL, 1.6 mmol) was added to asolution of (S)-methyl2-(4-(4-bromophenyl)-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-3-yl)-2-(tert-butoxy)acetate (83 mg, 0.16 mmol) in 1:1tetrahydrofuran/industrial methylated spirit (4 mL) at room temperature.The resulting mixture was heated to 60° C. and stirred at 60° C. 2hours. The resulting solution was concentrated in vacuo and the residuewas dissolved in water (10 mL). The aqueous solution was adjusted to pH5 by the addition of 2 M aqueous hydrochloric acid solution and theresulting suspension was extracted with dichloromethane (20 mL). Theorganic layer was washed with brine, dried (MgSO₄) and concentrated invacuo to give the crude product as a pale yellow solid. This waspurified by trituration with industrial methylated spirit (5 mL). Theresulting solid was collected by filtration to give the title compound(27 mg, 34%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ=1.02 (s, 9H),2.00-1.40 (m, 6H), 2.69 (s, 3H), 2.83-2.70 (m, 2H), 5.11 (s, 1H),7.25-7.06 (m, 1H), 7.63-7.42 (m, 3H). LCMS (run time=5 minutes, basic):R_(t)=3.19 minutes; m/z 490.00 [M+H⁺].

Antiviral Activity=0.080 μM (n=2) (S8737E).

HTRF Interaction assay=1160 nM (n=2) (S9118)

EXAMPLE 52Tert-Butoxy-(2-methyl-4-p-tolyl-5,8-dihydro-6H-7-oxa-9-thia-1-aza-fluoren-3-yl)-aceticacid

Step 1:(2-Amino-4,7-dihydro-5H-thieno[2,3-c]pyran-3-yl)-p-tolyl-methanone

4-Pyranone (2.31 mL, 24.97 mmol) and sulphur (801 mg, 24.97 mmol) wereadded to a solution of 3-oxo-3-p-tolyl-propionitrile (3.79 g, 23.8 mmol)in ethanol (40 mL) followed by morpholine (2.18 mL, 24.97 mmol) and thereaction heated at 40° C. for 16 hours, The reaction mixture wasconcentrated in vacuo and the residue purified by flash chromatographyon silica eluting with 0-20% ethylacetate/heptanes to afford12-Amino-4,7-dihydro-5H-thieno[2,3-c]pyran-3-yl)-p-tolyl-methanone as ayellow solid (2.2 g, 36% yield). LCMS (2 min acidic) 1.07 min ES+/AP+274 (MH+). ¹HNMR (400 MHz, CDCl₃) δ ppm 2.00-2.03 (m, 2H), 2.42 (s, 3H),3.80-3.84 (m, 2H), 4.80 (s, 2H), 6.60 (br s, 2H), 7.20 (d, 2H), 7.41 (d,2H).

Step 2:(2-Methyl-4-p-tolyl-5,8-dihydro-6H-7-oxa-9-thia-1-aza-fluoren-3-yl)-oxo-aceticacid ethyl ester

2,4-Dioxo-pentanoic acid ethyl ester (1.2 mL, 8.6 mmol) was added to asolution of2-Amino-4,7-dihydro-5H-thieno[2,3-c]pyran-3-yl)-p-tolyl-methanone (2.3g, 8.6 mmol) in ethanol (50 mL) followed by acetyl chloride (2.43 mL,34.3 mmol) and the reaction was warmed to 50° C. for 1 hour. Thereaction was then concentrated in vacuo and preadsorbed onto silica. Theresidue was purified by flash chromatography on silica eluting with0-40% ethyl acetate in heptanes to give the title compound as a redsolid (675 mg, 19%). LCMS (2 min acidic) 2.78 mins ES+/AP+ 396 (MH+).

Step 3:Hydroxy-(2-methyl-4-p-tolyl-5,8-dihydro-6H-7-oxa-9-thia-1-aza-fluoren-3-yl)-aceticacid ethyl ester

Sodium borohydride (97 mg, 2.56 mmol) was added to a stirred solution ofhydroxy-(2-methyl-4-p-tolyl-5,8-dihydro-6H-7-oxa-9-thia-1-aza-fluoren-3-yl)-aceticacid ethyl ester (675 mg, 1.71 mmol) in ethanol (20 mL). 2M HCl in water(3 mL) was added and then the reaction mixture concentrated in vacuo.The residue was partitioned between water (50 mL) and ethyl acetate (30mL). The layers were separated and the organics were dried (MgSO₄) andconcentrated in vacuo to afford the title compound as a cream solid (543mg, 80%) used without further purification. LCMS (2 min acidic) 1.20 minES+/AP+ 398. ¹HNMR (400 MHz, CDCl₃) δ ppm 1.20 (t, 3H), 1.82-1.88 (m,2H), 2.41 (s, 3H), 2.61 (s, 3H), 3.68-3.72 (m, 2H), 4.10-4.21 (m, 2H),4.81 (s, 2H), 7.16-7.26 (m, 4H).

Step 4:tert-Butoxy-(2-methyl-4-p-tolyl-5,8-dihydro-6H-7-oxa-9-thia-1-aza-fluoren-3-yl)-aceticacid ethyl ester

Tert-Butyl acetate (0.8 mL) was added to a solution ofhydroxy-(2-methyl-4-p-tolyl-5,8-dihydro-6H-7-oxa-9-thia-1-aza-fluoren-3-yl)-aceticacid ethyl ester (100 mg, 0.252 mmol) in dichloromethane (0.8 mL)followed by concentrated sulphuric acid (40 μL) and the reaction stirredat room temperature for 2 hours. Sodium bicarbonate solution (10%aqueous, 2 mL) was added and the reaction evaporated in vacuo until onlythe aqueous remained. The aqueous was extracted with ethyl acetate (2mL) and the organic layer washed with brine (1 mL), dried andconcentrated in vacuo. The residue was purified by flash chromatographyon silica eluting with 0-30% ethyl acetate/heptanes to afford acolourless solid (66 mg, 45%). LCMS (12 min acidic) 7.25 min, ES+/AP+454 (MH+)¹HNMR (400 MHz, CDCl₃) δ ppm 0.98 (s, 9H), 1.20 (t, 3H),1.55-1.65 (m, 2H), 2.43 (s, 3H), 2.77 (s, 3H), 3.57-3.61 (m, 1H),3.78-3.82 (m, 1H), 4.06-4.20 (m, 2H), 4.79-4.82 (m, 2H), 7.16 (d, 2H),7.32 (d, 2H).

Step 5:tert-Butoxy-(2-methyl-4-p-tolyl-5,8-dihydro-6H-7-oxa-9-thia-1-aza-fluoren-3-yl)-aceticacid

Aqueous sodium hydroxide (2N, 2 mL, 4 mmol) was added to a stirredsolution oftert-Butoxy-(2-methyl-4-p-tolyl-5,8-dihydro-6H-7-oxa-9-thia-1-aza-fluoren-3-yl)-aceticacid ethyl ester (66 mg, 0.15 mmol) in ethanol (2 mL) andtetrahydrofuran (2 mL) and the reaction was stirred for 2 hours at 60°C. The reaction was concentrated in vacuo until only the aqueousremained. The pH was adjusted to 2 by the addition of 2N aqueous HCl.The resulting solid was collected by filtration and purified bypreparative HPLC to afford the title compound. LCMS 3.63 min, ES+/AP+425.1 (MH+).

Column: Gemini-NX 3 μm C18 110A, ambient temperature; detection: UV 225nm—MS

Flow rate: 1.5 mL/min; mobile phase: A: H₂O+0.1% formic acid, B:MeCN+0.1% formic acid. Gradient (Time/mins, %B)—(0,5),(3,95),(4,95),(4.1,5),(5,5)

Antiviral Activity=0.189 μM (n=2) (S8737E).

HTRF Interaction assay=2750 nM (n=2) (S9118)

Evaluation of the Anti-HIV Activity of the Compounds of the InventionMT-2 Based Antiviral Assay (S8737E)

This assay is designed to determine the effects of small molecules onthe replication of HIV-1 in the lymphoblastoid cell line, MT2, and isable to detect the antiviral effect of compounds acting at any stage ofthe HIV-1 replication cycle. The assay, along with its associatedcytotoxicity assay, was described in detail in 2005 in a paper by Cao etal (Antimicrobial Agents and Chemotherapy 2005; 49(9), p 3833-3841).

MT2 cells are infected with the HIV-1 virus (NL4.3 strain; Adachi etal., Journal of Virology 1986) and transferred to assay platescontaining serial dilutions of compounds to be tested. The assay platesare incubated for 3 days (MT2) to allow for several rounds of viralreplication/infection to take place. At the end of this time,supernatant is transferred into new plates containing JC53BL cells.

JC53BL cells express CXCR4, CCR5 and CD4 receptors, and HIV-1-LTR—R-Gal.Under normal culture conditions undetectable levels of β-Galactosidaseare expressed, but in the presence of HIV-1, the viral Tat protein isable to activate the HIV-1-LTR in the JC53BL cells resulting in theincreased expression of the β-Gal enzyme. The expression can be measuredusing the ‘FluorAce β-Galactosidase reporter’ assay. The levels of β-Galare directly proportional to the levels of Tat (up to a threshold)allowing virus quantification. Compounds that inhibit virus replicationwill give rise to a reduced signal and a dose-response curve for eachcompound can be generated. This is then used to determine the IC50 foreach compound, a measure of the compound's potency.

The MT-2 based antiviral assay requires a separate cytotoxicityassessment of the compounds. This was performed using a 3 day MT-2cytotoxicity assay as follows:

3 Day MT2 Cytotoxicity Assay (S8738E)

The assay is designed to test whether or not compounds have cytotoxicactivity in MT2 cells by measuring the viability of these cells in thepresence of compounds. The assay is carried out by adding MT2 assayplates containing serial dilutions of the compounds to be screened.After 3 days incubation, the viability of the cells remaining in theplates is assayed using the commercially available reagent CellTiter-Glo(Promega Ltd). The data generated is then used to calculate theconcentration of compound required to cause 50% cytotoxicity (CC50).

All examples had CC50 >20 μM.

REFERENCES

-   Adachi, A., Gendelman, H., Koenig, S., Folks, T., Willey, R.,    Rabson, A. and Martin, M (1986) Production of acquired    immunodeficiency syndrome-associated retrovirus in human and    nonhuman cells transfected with an infectious molecular clone, J.    Virol., 59, 284-291.-   Cao J, Isaacson J, Patick A K, Blair W S. (2005) High-throughput    human immunodeficiency virus type 1 (HIV-1) full replication assay    that includes HIV-1 Vif as an antiviral target. Antimicrobial Agents    and Chemotherapy; 49(9), p 3833-3841.

Assay to Measure the LEDGF-Inteqrase Interation Inhibitory Activity ofCompounds of the Invention HTRF Interaction Assay (S9118)

An Homogeneous Time Resolved Fluorescence (HTRF) assay is performed in amanner similar to previous reports on HTRF protein protein assays asreviewed by Mathis (Clin. Chem., 2005). The assay procedure is performedas follows: reactions are performed in 20 μl final volume in 384-wellblack low volume microtiter plates (Greiner). The final reaction buffercontains 29 mM phosphase buffer (pH 7), 10 mM HEPES buffer (pH 7.4),68.5 mM NaCl, 1.4 mM KCl, 400 mM KF 0.05% (w/v) pluronic acid (P104,Sigma Aldrich) and 1% (v/v) DMSO. His₆-tagged integrase (78 nM finalconcentration) is incubated with mannose binding protein fused to theΔ325 carboxy terminal integrase binding domain of LEDGF in the presenceof compound for 2 hours at room temperature. Both these protein regentsare supplied by Prof. Zeger Debyser of Katholieke Universiteit Leuven,Leuven, Belgium. The compounds are added at varying concentrationsspanning a wide range from 0.1 up to 100 μM. Afterwards 8.3 nM ofeuropium cryptate conjugated anti-MBP monoclonal antibody and 17 nManti-His antibody conjugated with the acceptor fluorophore d2. Followinga 2 hour room temperature incubation the plates are read on an EnVision™microplate reader (Perkin Elmer) using an excitation wavelength of 320nM. The ratio of fluorescence emitted at 665 nM and 620 nM is used toassess the degree to which the protein-protein interaction had beeninhibited.

REFERENCES

-   Mathis G., Probing molecular interactions with homogeneous    techniques based on rare earth cryptates and fluorescence energy    transfer. Clin. Chem. 41 (9), 1391-7 (1995).

1. A compound of formula (I)

wherein: R¹ is CH₃, CH₂CH₃, Cl, Br, CHF₂ or CF₃; R² is H, OH or F; X isCH₂ or O; provided that when R¹ is CH₃ and R² is H, X is O; or apharmaceutically acceptable salt thereof.
 2. A compound according toclaim 1 selected from:

or a pharmaceutically acceptable salt thereof.
 3. A compound accordingto claim 1 of formula (Ia)

wherein: R¹ is CH₃, CH₂CH₃, Cl, Br, CHF₂ or CF₃; R² is H, OH or F; X isCH₂ or O; provided that when R¹ is CH₃ and R² is H, X is O; or apharmaceutically acceptable salt thereof.
 4. A compound according toclaim 1 selected from:

or a pharmaceutically acceptable salt thereof.
 5. A compound accordingto claim 1 selected from:

or a pharmaceutically acceptable salt thereof.
 6. A pharmaceuticalcomposition comprising a compound as claimed in claim 1 together with apharmaceutically acceptable excipient.
 7. A method of treatment of amammal, including a human being, to treat HIV infection, comprisingadministering to said mammal an effective amount of a compound asclaimed in claim
 1. 8. A compound as claimed in claim 1 in combinationwith one or more other pharmacologically active agents.
 9. A compound asclaimed in claim 1 in combination with one or more other agents whichare useful for the treatment of HIV infection.
 10. A product comprisinga compound as claimed in claim 1 and one or more other pharmacologicallyactive agents as a combined preparation for simultaneous, separate orsequential use in therapy.
 11. A kit comprising two or morepharmaceutical compositions, at least one of which comprises a compoundas claimed in claim 1 together with a pharmaceutically acceptableexcipient, and means for separately retaining said compositions.